SIRT1-mediated FoxOs pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride

Abstract

Fluorine may result in damage to teeth, bones and other body tissues, and is a serious public health problem. SIRT1 deacetylates FOXOs, which brings about apoptosis and autophagy promotion or suppression. Fluorine may induce cell apoptosis, however, the role of autophagy in apoptosis induced by fluorine is still poorly understood, and the interaction between SIRT1 and FOXOs should be further illustrated. Therefore, this study investigated the mechanisms underlying the NaF- induced apoptosis and autophagy in osteoblast-like MC3T3-E1 cells in vitro through activating or inhibiting SIRT1. Via RT-PCR, western blot, flow cytometry assays, fluorescence and laser confocal microscopy, it was found that NaF induced both cell apoptosis and autophagy. Results also showed that NaF up-regulated SIRT1 expression in a dose-dependent manner. The autophagy of MC3T3-E1 was also up- regulated indirectly whilst apoptosis was significantly attenuated when incubated with the SIRT1 activator SRT1720. When SIRT1 inhibitor Ex-527 was used, the latter effects were reversed. Furthermore, SIRT1 increased deacetylation of FoxO1 and promoted the up-regulation of its target substrate Rab7, as well as increase of Bnip3 which was substrate of FoxO3, and we hypothesize that these pathways may cause an increase in autophagic flux and a reduction in apoptosis. In conclusion, SIRT1-induced autophagy enhancement protects against fluoride-induced apoptosis through autophagy induction in MC3T3-E1 cells, which may be associated with a SIRT1-FoxO1-Rab7 axis and a SIRT1-FoxO3-Binp3 axis. The role of SIRT1 in selecting between cell survival and death provides a potential therapeutic strategy in fluorosis.

Keywords: MC3T3-E1 cells; SIRT1; apoptosis; autophagy; sodium fluoride.

Figure 1. Assessment of apoptosis in cells treated with 10^–6, 10^–5, 10^–4 and 10^–3 mol/L NaF

(A) The apoptotic rates were detected by FCM of annxin V-FITC/PI dual staining. Q1 quadrant (annexin V–, PI+) represented dead cells; Q2 quadrant (annexin V+, PI+) represented late apoptotic cells; Q3 quadrant (annexin V+, PI–) represented early apoptotic cells; Q4 quadrant (annexin V–, PI–) represented live cells. (B) The caspase 3 mRNA levels were detected using RT-PCR assay. Columns, mean of three independent experiments; mean ± SD; ^*P < 0.05,^{* *}P < 0.01; ^#P < 0.05; ^##P < 0.01. The same as below.

Figure 2. Assessment of autophagy in osteoblast induced by NaF

(A) The LC3 mRNA levels were detected using RT-PCR assay. β-actin was used as an reference gene. (B) The Beclin1 mRNA levels were detected using RT-PCR assay. (C) The LC3-II, LC3-I protein levels of treated cells were detected using Western blot assay. (D) Results of densitometric analysis for Western blot.

Figure 3. Determination of SIRT1 in osteoblast induced by NaF

After the cells reached a steady-state of exponential growth in normal media, they were exposed to different concentrations of sodium fluoride for indicated time. And the SIRT1 mRNA levels were detected using RT-PCR assay.

Figure 4. Effects of SIRT1 on cell viability of MC3T3-E1 exposed to NaF

MC3T3-E1 cells were pretreated with SRT1720 or Ex-527 or CQ at indicated concentrations for 2 h, and then treated with 10^–4 M NaF for 6 h, 12 h and 24 h. Viable cells were detected by MTT assay and cell viability (%) was calculated. The data were represented as mean ± SD from three independent experiments. Note: SF1: SRT1720 (100 nmol/L)+NaF, SF2: SRT1720 (200 nmol/L)+NaF, EF1: Ex-527 (400 nmol/L)+NaF,EF2: Ex-527 (800 nmol/L)+NaF, CF1: CQ (50 nmol/L)+NaF, CF2: CQ (50 nmol/L)+NaF.

Figure 5. Effects of autophagy on cell viability of MC3T3-E1 exposed to NaF

MC3T3-E1 cells were pretreated with Rapamycin or SRT1720 or SRT1720 combined with 3-MA at indicated concentrations for 2 h, and then treated with 10^–4 M NaF for 48 h. Viable cells were detected by MTT assay and cell viability (%) was calculated. The data were represented as mean ± SD from three independent experiments. Note: RF1: Rapamycin (100 nmol/L)+NaF, RF2: Rapamycin (200 nmol/L)+NaF, SF: SRT1720 (200 nmol/L)+NaF, SMF1: SRT1720 (200 nmol/L)+3-MA (5 mmol/L)+NaF, SMF2: SRT1720 (200 nmol/L)+3-MA (10 mmol/L)+NaF.

Figure 6. SIRT1 inhibited NaF-induced apoptosis

FCM of annxin V-FITC/PI dual staining was performed and cleaved-caspase 3 protein expression level was detected to evaluate apoptosis in cells treated with NaF alone, pre-incubated with SRT1720 or pre-incubated with Ex-527. (A) The apoptotic rates were detected by FCM of annxin V-FITC/PI dual staining. (B) The cleaved-caspase 3 expression levels were detected using Western blot assay. (C) Results of densitometric analysis for Western blot.

Figure 7. SIRT1 enhanced NaF-induced autophagy

The LC3 protein and Beclin1 mRNA levels were detected in cells treated with NaF alone, pre-incubated with SRT1720 or pre-incubated with Ex-527 or pre-incubated with CQ. (A) and (B) After designed experiment measures, the LC3 and Beclin1 mRNA levels were detected using RT-PCR assay. (C) The LC3 protein levels of treated cells were detected using Western blot assay. (D) Results of densitometric analysis for Western blot.

Figure 8. Fluorescence microscope analysis was performed to evaluate autophagy in cells treated with NaF alone, pre-incubated with SRT1720 or pre-incubated with Ex-527

(A–D) (200×). Localization of LC3-II at autophagosome membrane was indicated as green fluorescent punctate dots. The representative images are shown.

Figure 9. Confocal microscope analysis was performed to evaluate autophagy in cells treated with NaF alone, pre-incubated with SRT1720 or pre-incubated with Ex-527

(A–D) (400×). Representative images of fluorescent LC3 puncta after Ad-tf-LC3 transduction are shown.

Figure 10. The pathways mediated by SIRT1 in NaF-induced apoptosis and autophagy

The protein expression levels of FoxO1 and Ac-FoxO1 were detected to evaluate SIRT1-mediated pathways in cells treated with NaF alone, pre-incubated with SRT1720 or pre-incubated with Ex-527. (A, B) The FoxO1 and Ac-FoxO1 expression levels were detected using Western blot assay. (C, D) Results of densitometric analysis for Western blot.

Figure 11. The pathways mediated by SIRT1 in NaF-induced apoptosis and autophagy

The protein expression levels of Rab7 and Binp3 were detected to evaluate SIRT1-mediated pathways in cells treated with NaF alone, pre-incubated with SRT1720 or pre-incubated with Ex-527. (A, B) The Rab7 and Binp3 expression levels were detected using Western blot assay. (C, D) Results of densitometric analysis for Western blot.

Figure 12. Positive regulation of FoxO factors by oxidative stress stimuli and the proposal signaling pathway of the current study

(A) Oxidative stress induces the phosphorylation, acetylation and monoubiquitination of FoxO factors at a number of regulatory sites by several factors. In response to oxidative stress, FoxO factors translocate to the nucleus and bind to the deacetylase SIRT1, and specific genes involved in cell-cycle arrest and the response to stress are recruited. p27, cyclin-dependent kinase inhibitor; MnSOD, manganese superoxide dismutase; Bim, pro-apoptotic Bcl2-interacting mediator of cell death; Gadd45α, growth arrest- and DNA damage-inducible gene 45 α. (Dervis A M Salih and Anne Brunet./Curr Opin Cell Biol. 2008; 20(2): 126–136). (B) Fluoride induces ROS generation resulting in cell apoptosis. Meanwhile, fluoride activates SIRT1 through the JNK/c-Jun pathway. Activation of SIRT1 protects against fluoride-induced osteoblast apoptosis probably through two following mechanisms: SIRT1-FoxO1-Rab7 axis and SIRT1-FoxO3-Binp3 axis via autophagy promotion.