SIRT1 Regulates PTEN/Akt/FOXO1 Pathway to Trigger ROS-Induced Apoptosis In Mouse Embryonic Stem Cells.

Abstract 1610

Embryonic stem (ES) cells have potential usefulness for regenerative therapy due to their self-renewal and pluripotency properties. SIRT1, an NAD⁺-dependent histone deacetylase, plays a critical role in endogenous reactive oxygen species (ROS)-triggered apoptosis in mouse ES (mES) cells by blocking nuclear translocation of p53 (Han eta al., Cell Stem Cell, 2008). Here, we investigated a possible role for phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/Akt pathway in the SIRT1-mediated apoptosis pathway in mES cells. SIRT1 plays a role in apoptosis and stress resistance by deacetylating important proteins including p53, NF-κB, Ku70, PTEN and FOXO transcription factors. Since the PI3K/Akt pathway plays an important role in protecting cells from stresses including ROS, we investigated whether the PI3K/Akt pathway was involved in SIRT1-mediated apoptosis in mES cells. Anti-oxidant free culture condition suppressed Akt phosphorylation in wild type (WT) mES cells, and phosphorylation of Akt was enhanced by removal of 2-mercaptoethanol (2-ME) in SIRT1^-/- cells. Since PTEN is a negative regulator of Akt and its activity can be modulated by acetylation, we investigated if SIRT1 deacetylated PTEN to down-regulate Akt to trigger apoptosis in anti-oxidant-free culture conditions. PTEN protein levels were not changed after 2-ME withdrawal. However, PTEN was sequestered in the cytoplasm in SIRT1^-/- cells after removal of 2-ME, consistent with increased Akt phosphorylation. Acetylation of PTEN has been reported to regulate its phosphatase activity. We evaluated whether SIRT1 deacetylated PTEN to regulate Akt activity in response to endogenous ROS. SIRT1-deficiency enhanced basal acetylation levels of PTEN and sustained acetylation levels after 2-ME withdrawal in SIRT1^-/- cells. Nuclear PTEN was also hyper-acetylated in SIRT1^-/- cells. This suggests that SIRT1 deacetylates PTEN to prevent Akt activation after removal of 2-ME, which in turn initiates apoptosis in response to ROS. As SIRT1 and Akt regulate FOXO1 transcription factor, we investigated whether the SIRT1-Akt-FOXO1 pathway was responsible for apoptosis in response to endogenous ROS. SIRT1-deficiency enhanced the acetylation/phosphorylation level of FOXO1 and subsequently inhibited the nuclear localization of FOXO1. Dephosphorylation and activation of FOXO induces expression of proapoptotic BH-3 only proteins, Bim and Puma to trigger apoptosis in response to ROS. BH3-only proteins initiate the mitochondrial apoptotic pathway in response to a variety of stresses by modulating multi-domain Bcl-2 family proteins. Bim protein level was significantly increased 24h after culture in 2-ME-free media in WT cells, but not by treatment of cells with DNA damaging agent, mitomycin C (MMC). In SIRT1^-/- cells, Bim protein level was not enhanced by MMC treatment and 2-ME withdrawal. Puma α was also induced in WT mES cells by MMC treatment as well as 2-ME withdrawal. In SIRT1^-/- mES cellst, Puma α was induced only by MMC treatment, not by 2-ME withdrawal. Bax expression was unaffected by 2-ME withdrawal or MMC treatment. Thus, we found that Bim and Puma expression is induced by endogenous ROS in a SIRT1-dependent manner. Caspase 3, a key mediator of apoptosis in mammalian cells, was activated by both 2-ME withdrawal and MMC treatment in WT cells, but only by MMC treatment in SIRT1^-/- cells. In summary, we found that SIRT1 plays a critical role in regulation of the PTEN/Akt/FOXO1 pathway to modulate apoptosis in response to ROS in mES cells. Our data support an important role of SIRT1 in PTEN/Akt/FOXO1-mediated Bim and Puma expression to discard damaged mES cells.