SIRT1 deficiency downregulates PTEN/JNK/FOXO1 pathway to block reactive oxygen species-induced apoptosis in mouse embryonic stem cells

Abstract

Silent mating type information regulation 2 homolog 1 (SIRT1) plays a critical role in reactive oxygen species-triggered apoptosis in mouse embryonic stem (mES) cells. Here, we investigated a possible role for the PTEN/Akt/JNK pathway in the SIRT1-mediated apoptosis pathway in mES cells. Akt was activated by removal of anti-oxidant 2-mercaptoethanol in SIRT1(-/-) mES cells. Since PTEN is a negative regulator of Akt and its activity can be modulated by acetylation, we investigated if SIRT1 deacetylated PTEN to downregulate Akt to trigger apoptosis in anti-oxidant-free culture conditions. PTEN was hyperacetylated and excluded from the nucleus in SIRT1(-/-) mES cells, consistent with enhanced Akt activity. SIRT1 deficiency enhanced the acetylation/phosphorylation level of FOXO1 and subsequently inhibited the nuclear localization of FOXO1. Cellular acetylation levels were enhanced by DNA-damaging agent, not by removal of anti-oxidant. c-Jun NH2-terminal kinase (JNK) was activated by removal of anti-oxidant in SIRT1-dependent manner. Although p53 acetylation was stronger in SIRT1(-/-) mES cells, DNA-damaging stress activated phosphorylation and enhanced cellular levels of p53 irrespective of SIRT1, whereas removal of anti-oxidant slightly activated p53 only with SIRT1. Expression levels of Bim and Puma were increased in anti-oxidant-free culture conditions in an SIRT1-dependent manner and treatment with JNK inhibitor blocked induction of Bim expression. DNA-damaging agent activated caspase3 regardless of SIRT1. Our data support an important role for SIRT1 in preparing the PTEN/JNK/FOXO1 pathway to respond to cellular reactive oxygen species.

FIG. 1.

AKT is activated by 2-ME withdrawal from SIRT1^−/− mES cells. (A) ES cells were cultured with or without 2-ME (100 μM, 24 h). Apoptotic cells were assessed by AnnexinV/7-AAD staining. Data represent mean ± SEM (n = 3). (B) ES cells were cultured with or without 2-ME, and then total lysates (upper panel) and nuclear lysates (lower panel) were extracted. Phospho-AKT, AKT, and PTEN expression were measured by immunoblotting. Anti-lamin B and anti β-tubulin antibodies were used as loading controls. Relative expression levels are presented as fold induction above expression levels in nontreated control cells. Data are graphed as mean ± SEM (n = 3), except for nuclear Akt phosphorylation data (n = 4). (C) Total extracts and nuclear extracts of mES cells were immunoprecipitated using anti-PTEN antibody. Immunoprecipitation products were analyzed by immunoblotting for measuring acetylation of PTEN. Relative PTEN acetylation level (the value of control WT mES cells is set as 1) is shown. Data are graphed as mean ± SEM (total extracts, n = 5; nuclear extracts, n = 3). *P < 0.05, **P < 0.01. Representative blots of at 3 different experiments are shown in parts (B) and (C). mES, mouse embryonic stem; WT, wild-type; 2-ME, 2-mercaptoethanol; SIRT1, silent mating type information regulation 2 homolog 1.

FIG. 2.

Nuclear import of FOXO1 is induced by removal of 2-ME in an SIRT1-dependent manner. WT and SIRT1^−/− mES cells were cultured with or without 2-ME (100 μM, 12 h). Total extracts (A) and nuclear extracts (B) were prepared. (A) FOXO1 expression, phosphorylation, and acetylation were assessed by immunoblotting using anti-FOXO1, anti-phospho FOXO1 (Thr32), and anti-acetylated FOXO1 antibodies. The relative expression levels (the value of control WT mES cells is set as 1) are shown. (B) Nuclear FOXO1 was measured by immunoblotting using anti-FOXO1 antibody. Relative protein levels of nuclear FOXO1 (the value of each control ES cells is set as 1) are shown. Anti-lamin B and anti β-tubulin antibodies were used as loading controls. Each values represent as mean ± SEM (total extracts, n = 5; nuclear extracts, n = 3). *P < 0.05.

FIG. 3.

Removal of 2-ME enhances JNK phosphorylation, not cellular acetylation. WT and SIRT1^−/− mES cells were cultured with or without 2-ME (100 μM) for 24 h or treated with 2-ME and MMC (1 μg/mL) for 6 h, and then total extracts were prepared. (A) Total acetylated proteins were detected by using anti-acetylated lysine antibody. (B) JNK phosphorylation was assessed by immunoblotting using anti-phospho-JNK antibody. Relative phospho-JNK levels are presented as fold induction above expression levels in nontreated control cells. Values are shown as mean ± SEM (n = 3). **P < 0.01. JNK, c-Jun NH2-terminal kinase; MMC, mitomycin C.

FIG. 4.

Differential regulation of p53 by ROS and DNA-damaging stress. WT and SIRT1^−/− mES cells were cultured with or without 2-ME (100 μM) for 24 h or treated with 2-ME and MMC (1 μg/mL) for 6 h, and then total extracts were prepared. Total p53 protein, and phospho and acetylated p53 were detected by immunoblotting. Relative expression levels (the value of control WT mES cells is set as 1) are shown. Values represent mean ± SEM (n = 3). *P < 0.01; **P < 0.01. ROS, reactive oxygen species.

FIG. 5.

Removal of 2-ME induces expression of Bim and Puma to activate Caspase3 through JNK. (A) WT and SIRT1^−/− mES cells were cultured with or without 2-ME (100 μM) for 24 h or treated with 2-ME and MMC (1 μg/mL) for 6 h. Cells were harvested and expression levels of proapototic Bcl2-family proteins were assessed by immunoblotting. Relative expression levels of each protein are presented as fold induction above expression levels in nontreated control cells. Values are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01. (B) WT mES cells treated with JNK inhibitor SP600125 (10 μM) in the presence or absence of 2-ME (100 μM) for 24 h. Total lysates were prepared, and then Bim EL expression level was assessed by immunoblotting. Relative expression levels of Bim EL (the value of control WT mES cells is set as 1) are shown. Data are graphed as mean ± SEM (n = 3). **P < 0.01.

FIG. 6.

Schematic diagram of SIRT1 function in ROS-induced apoptosis in mES cells. SIRT1 is required for ROS-induced JNK activation, which results in activation and nuclear translocation of FOXO1 to induce Bim/Puma expression. SIRT1 also positively regulates FOXO1 by direct deacetylation and via inactivation of Akt. Together with p53, Bim/Puma initiates mitochondrial-mediated apoptosis.