The stabilization of hypoxia inducible factor modulates differentiation status and inhibits the proliferation of mouse embryonic stem cells

Chem Biol Interact. 2016 Jan 25;244:204-14. doi: 10.1016/j.cbi.2015.12.007. Epub 2015 Dec 23.

Abstract

Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells.

Keywords: DMOG; Embryonic stem cells; HIF-1; Hypoxia; JNJ-42041935; Prolyl hydroxylase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids, Dicarboxylic / chemistry
  • Amino Acids, Dicarboxylic / pharmacology
  • Animals
  • Benzimidazoles / chemistry
  • Benzimidazoles / pharmacology
  • Cell Cycle / drug effects
  • Cell Differentiation / drug effects*
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Hypoxia / metabolism
  • Hypoxia-Inducible Factor 1 / metabolism*
  • Hypoxia-Inducible Factor-Proline Dioxygenases / antagonists & inhibitors
  • Hypoxia-Inducible Factor-Proline Dioxygenases / metabolism
  • Mice
  • Mouse Embryonic Stem Cells / cytology*
  • Mouse Embryonic Stem Cells / drug effects*
  • Mouse Embryonic Stem Cells / metabolism
  • Protein Stability / drug effects
  • Pyrazoles / chemistry
  • Pyrazoles / pharmacology
  • Structure-Activity Relationship

Substances

  • 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid
  • Amino Acids, Dicarboxylic
  • Benzimidazoles
  • Enzyme Inhibitors
  • Hypoxia-Inducible Factor 1
  • Pyrazoles
  • Hypoxia-Inducible Factor-Proline Dioxygenases
  • oxalylglycine