Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3β Signaling Pathway and Promotes Hepatocellular Injury

Abstract

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Keywords: endoplasmic reticulum stress (ER stress); gene regulation; liver injury; phosphatidylinositol signaling; sirtuin 1 (SIRT1).

FIGURE 1.

ER stress increases the protein expression of SIRT1 in vitro. A, A549 cells were treated with 1 μm TG for the indicated time. B and C, relative protein quantity of SIRT1 (B) and GRP78 (C) was analyzed by Image Gauge software and normalized with γ-tubulin. Values are the mean ± S.E. (error bars) (n = 3 for B and C). *, p < 0.05 versus control as assessed by one-way analysis of variance with Dunnett procedure. D and E, A549 cells were treated with the indicated concentrations of TG for 24 h (D) or with TM for 12 h (E). THP-1 cells (F) and HEK293 cells (G) were treated for 12 h with TG (1 μm). H, mouse embryonic fibroblast (MEF) cells were treated for 24 h with TG (1 μm). Expression of SIRT1 and GRP78 protein was determined by Western blotting analysis of whole cell lysate. γ-Tubulin was used as a loading control. CON, control.

FIGURE 2.

ER stress increases the protein expression of SIRT1 in vivo. A–C, C57BL/6J mice were intraperitoneally injected with TM (1 μg/g of body weight), and liver tissues were harvested at 4, 8, or 24 h after injection. Expression of mSIRT1 and mGRP78 protein was determined by Western blotting analysis of whole cell lysate. Mouse γ-tubulin (mγ-tubulin) was used as a loading control. The relative quantity of mSIRT1 (B) and mGRP78 (C) protein was analyzed by Image Gauge software. Values are the mean ± S.E. (error bars) (n = 3 for B and C). *, p < 0.05 versus control; ***, p < 0.001 versus control; †, p < 0.1 versus control as assessed by Student's t test. n.s., not significant. D, immunohistochemical analysis of SIRT1 in TM-injected mouse liver tissue. Livers were collected 24 h after intraperitoneal injection with TM. Rabbit IgG was used as a negative control. Scale bars indicate 20 μm. CON, control.

FIGURE 3.

ER stress increases the expression of SIRT1 at transcriptional level. A and B, A549 cells were treated with TG (1 μm) (A) or with TM (5 μg/ml) (B) for the indicated time. The relative quantity of SIRT1 mRNA was analyzed by real time quantitative RT-PCR. 18S rRNA was used as an internal control. C, mice were administered intraperitoneally with TM (1 μg/g of body weight), and liver tissues were harvested at the indicated time after injection. The relative quantity of mouse SIRT1 mRNA was analyzed by real time quantitative RT-PCR using mouse 18S rRNA as an internal control. Values are the mean ± S.E. (error bars) (n = 3 for A and B). *, p < 0.05; **, p < 0.01 as assessed by Student's t test. n.s., not significant; CON, control.

FIGURE 4.

XBP1, ATF4, and ATF6 are likely dispensable for ER stress-induced SIRT1 up-regulation. A, A549 cells were transfected with SIRT1 promoter plasmids (−1200, −691, −457, or −47 bp) and treated with 1 μm TG for 24 h. Luciferase activity was determined 48 h after plasmid transfection and is expressed as -fold activation over untreated samples. B–D, A549 cells were transfected with 0.1 or 1.0 μg XBP1s (B), ATF4 (C), or ATF6α(1–373) (D). E–G, A549 cells were transfected with siRNA for XBP1 and/or ATF4. Forty-eight hours post-transfection, cells were treated with TG (1 μm) for 3 h. The relative quantity of the indicated gene was analyzed by real time quantitative RT-PCR. 18S rRNA was used as an internal control. Values are the mean ± S.E. (error bars) (n = 3 for A–G). *, p < 0.05; **, p < 0.01; ***, p < 0.001 as assessed by Student's t test. n.s., not significant; CON, control.

FIGURE 5.

ER stress-induced SIRT1 up-regulation is mediated by the PI3K-Akt-GSK3β signaling pathway. A, A549 cells were transfected with SIRT1 promoter plasmid (−457 bp). Cells were pretreated with inhibitors SB203580 (SB; 20 μm), CHIR99021 (CHIR; 2 μm), SP600125 (SP; 20 μm), and wortmannin (WM; 1 μm) for 1 h and co-treated with TG (1 μm) for 24 h. Luciferase activity was determined 48 h after plasmid transfection and is expressed as -fold activation over TG-untreated samples. B, A549 cells were treated with 1 μm TG for the indicated time and subjected to Western blotting analysis. β-Actin was used as a loading control. C–F, A549 cells were pretreated with SP600125 (C), wortmannin (D), CHIR99021 (E), and LiCl (F) for 1 h and treated with TG for 3 h. G and H, A549 cells were transfected with siRNA for GSK3β. Forty-eight hours after transfection, cell lysates were subjected to Western blotting (G) or TG treatment for QPCR analysis (H). Relative SIRT1 mRNA expression was analyzed by QPCR analysis. 18S rRNA was used as an internal control. *, p < 0.05; **, p < 0.01; ***, p < 0.001 as assessed by Student's t test (A) and by one-way analysis of variance with Bonferroni test (C–F and H). Values are the mean ± S.E. (error bars) (n = 3 for A and C–F). n.s., not significant; CON, control; NT, no treatment; p-Akt, phospho-Akt; p-GSK3β, phospho-GSK3β.

FIGURE 6.

ER stress-induced cell death is positively regulated by SIRT1. A, A549 cells were treated with TG (1 μm) for 24 h with or without EX527, a SIRT1 inhibitor (10 μm). The relative -fold increase of cell mortality was assessed by lactate dehydrogenase (LDH) assay. B–E, mice were injected with TM (1 μg/g) and EX527 (2 μg/g) on Day 0 and Day 1. Livers were collected 72 h after the first injection and subjected to H&E staining (B), hepatic injury quantification (C), and real time quantitative PCR analysis (D and E). 18S rRNA was used as an internal control. Values are the mean ± S.E. (error bars) (n = 3–4 for A, C, and D). Scale bars indicate 20 μm (B). Right panels are high magnification photos of middle panels (B). *, p < 0.05; **, p < 0.01 as assessed by analysis of variance with Dunnett procedure. n.s., not significant. F, TM- and TM + EX527-treated mouse livers were subjected to immunohistochemistry for acetylated p53 (upper panels) and acetylated p65 (bottom panels). Scale bars indicate 20 μm. CON, control; Veh, vehicle.

FIGURE 7.

Schematic diagram of how ER stress regulates SIRT1 expression. The up-regulation of SIRT1 expression by ER stress is not through the conventional ER stress pathways but rather through the PI3K-Akt-GSK3β signaling pathway. The ER stress-induced SIRT1 leads to hepatocellular damage.