SIRT1 positively regulates autophagy and mitochondria function in embryonic stem cells under oxidative stress

Xuan Ou, Man Ryul Lee, Xinxin Huang, Steven Messina-Graham, Hal Broxmeyer

Research output: Contribution to journalArticle

130 Citations (Scopus)

Abstract

SIRT1, an NAD-dependent deacetylase, plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress, and has been linked to age-related reactive oxygen species (ROS) generation, which is highly dependent on mitochondrial metabolism. H2O 2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H 2O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-, than in WT mESCs. However, addition of 3-methyladenine, a widely used autophagy inhibitor, in combination with H 2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II, lowered expression of Beclin-1, and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs, suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs, inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress, effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways.

Original languageEnglish
Pages (from-to)1183-1194
Number of pages12
JournalStem Cells
Volume32
Issue number5
DOIs
StatePublished - 2014

Fingerprint

Autophagy
Embryonic Stem Cells
Mitochondria
Oxidative Stress
Reactive Oxygen Species
Mitochondrial Dynamics
Apoptosis
70-kDa Ribosomal Protein S6 Kinases
S 6
Lentivirus
Mitochondrial Membrane Potential
Mouse Embryonic Stem Cells
Phosphatidylinositol 3-Kinases
NAD
Small Interfering RNA
Cell Death
Phosphorylation
Staining and Labeling

Keywords

  • Apoptosis
  • Autophagy
  • Cell culture
  • Cell signaling
  • Embryonic stem cells
  • Oxidative stress
  • Sirt1

ASJC Scopus subject areas

  • Cell Biology
  • Developmental Biology
  • Molecular Medicine
  • Medicine(all)

Cite this

SIRT1 positively regulates autophagy and mitochondria function in embryonic stem cells under oxidative stress. / Ou, Xuan; Lee, Man Ryul; Huang, Xinxin; Messina-Graham, Steven; Broxmeyer, Hal.

In: Stem Cells, Vol. 32, No. 5, 2014, p. 1183-1194.

Research output: Contribution to journalArticle

Ou, Xuan ; Lee, Man Ryul ; Huang, Xinxin ; Messina-Graham, Steven ; Broxmeyer, Hal. / SIRT1 positively regulates autophagy and mitochondria function in embryonic stem cells under oxidative stress. In: Stem Cells. 2014 ; Vol. 32, No. 5. pp. 1183-1194.
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AB - SIRT1, an NAD-dependent deacetylase, plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress, and has been linked to age-related reactive oxygen species (ROS) generation, which is highly dependent on mitochondrial metabolism. H2O 2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H 2O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-, than in WT mESCs. However, addition of 3-methyladenine, a widely used autophagy inhibitor, in combination with H 2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II, lowered expression of Beclin-1, and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs, suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs, inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress, effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways.

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