Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling

J Obstet Gynaecol Res. 2017 Oct;43(10):1585-1596. doi: 10.1111/jog.13410. Epub 2017 Jul 10.


Aim: Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts.

Methods: HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively.

Results: Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1.

Conclusion: mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling.

Keywords: O-GlcNAc; apoptosis; autophagy; mTOR; phosphorylation.

MeSH terms

  • Apoptosis
  • Autophagy / physiology*
  • Beclin-1 / metabolism*
  • Cell Line
  • Humans
  • N-Acetylglucosaminyltransferases / metabolism*
  • Qb-SNARE Proteins / metabolism*
  • Qc-SNARE Proteins / metabolism*
  • Signal Transduction / physiology*
  • TOR Serine-Threonine Kinases / antagonists & inhibitors
  • TOR Serine-Threonine Kinases / metabolism*
  • Trophoblasts / metabolism*


  • Beclin-1
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • SNAP29 protein, human
  • N-Acetylglucosaminyltransferases
  • UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase
  • MTOR protein, human
  • TOR Serine-Threonine Kinases