Diverse actions of sirtuin-1 on ovulatory genes and cell death pathways in human granulosa cells

Abstract

Background: Human granulosa-lutein cells (hGLCs) amply express sirtuin-1 (SIRT1), a NAD + -dependent deacetylase that is associated with various cellular functions. SIRT1 was shown to elevate cAMP on its own and additively with human chorionic gonadotropin (hCG), it is therefore interesting to examine if SIRT1 affects other essential hGLC functions.

Methods: Primary hGLCs, obtained from the follicular aspirates of women undergoing IVF and SV40-transfected, immortalized hGLCs (SVOG cells), were used. Primary cells were treated with SIRT1 specific activator SRT2104, as well as hCG or their combination. Additionally, siRNA-targeting SIRT1 construct was used to silence endogenous SIRT1 in SVOG cells. PTGS2, EREG, VEGFA and FGF2 expression was determined using quantitative polymerase chain reaction (qPCR). Apoptotic and necroptotic proteins were determined by specific antibodies in western blotting. Cell viability/apoptosis was determined by the XTT and flow cytometry analyses. Data were analyzed using student t-test or Mann-Whitney U test or one-way ANOVA followed by Tukey HSD post hoc test.

Results: In primary and immortalized hGLCs, SRT2104 significantly upregulated key ovulatory and angiogenic genes: PTGS2, EREG, FGF2 and VEGFA, these effects tended to be further augmented in the presence of hCG. Additionally, SRT2104 dose and time-dependently decreased viable cell numbers. Flow cytometry of Annexin V stained cells confirmed that SIRT1 reduced live cell numbers and increased late apoptotic and necrotic cells. Moreover, we found that SIRT1 markedly reduced anti-apoptotic BCL-XL and MCL1 protein levels and increased cleaved forms of pro-apoptotic proteins caspase-3 and PARP. SIRT1 also significantly induced necroptotic proteins RIPK1 and MLKL. RIPK1 inhibitor, necrostatin-1 mitigated SIRT1 actions on RIPK1 and MLKL but also on cleaved caspase-3 and PARP and in accordance on live and apoptotic cells, implying a role for RIPK1 in SIRT1-induced cell death. SIRT1 silencing produced inverse effects on sorted cell populations, anti-apoptotic, pro-apoptotic and necroptotic proteins, corroborating SIRT1 activation.

Conclusions: These findings reveal that in hGLCs, SIRT1 enhances the expression of ovulatory and angiogenic genes while eventually advancing cell death pathways. Interestingly, these seemingly contradictory events may have occurred in a cAMP-dependent manner.

Keywords: Angiogenesis; Apoptosis; Necroptosis; Ovulation.

Fig. 1

SRT2104 augments ovulatory and angiogenic genes in SVOG cells. Cells were incubated with either control media (designated as 1) or SRT2104 (50 μmol/L) for 24 h. Cells were then harvested, and mRNA expression was determined using qPCR. The results are presented as the means ± SEM of 3 independent experiments. Asterisks indicate significant (**p < 0.01 and ***p < 0.001) statistical differences from the control

Fig. 2

SRT2104 augments ovulatory and angiogenic genes in primary hGLCs. Cells were incubated with control medium alone, hCG (10 IU), SRT2104 (50 μmol/L), or the combined treatment of hCG and SRT2104 for 24 h. Cells were then harvested, and mRNA expression was determined using qPCR. The results are presented as the means ± SEM of 4 independent experiments. The different letters indicate significant statistical differences

Fig. 3

SRT2104 decreased viable cell numbers in a dose and time—dependent manner. SVOG cells were incubated with either control media or with varying concentrations (10, 25 or 50 μmol/L) of SRT2104 for 24, 48 and 72 h. Cell viability was determined using XTT assay. The results are presented as the means ± SEM of 3 independent experiments. The different letters indicate significant statistical differences at p < 0.05 analyzed using ANOVA followed by Tukey HSD post-hoc multiple comparison test

Fig. 4

SRT2104 reduces live cells and increases late apoptotic and necrotic cells. A and B SVOG cells were treated with either control media or SRT2104 (50 μmol/L) or C and D cells were transfected with 10 nmol/L of either scrambled siRNA (siNC) or SIRT1 siRNA (siSIRT1). After 48 h post- treatment or transfection, cells were analyzed using FACS after propidium iodide (PI) and Annexin V staining (A and C). Results are presented as the means ± SEM from 4 and 3 independent experiments for SRT2104-treated and siSIRT1 transfected cells, respectively. E Comparison of mean cell populations between SIRT1 silencing and activation. The mean difference was calculated by subtracting the mean cell population of SRT2104- or siSIRT1- treated cells from their respective controls. Asterisks (**p < 0.01, ***p < 0.001) indicate significant differences between cells treated with SRT2104 or siSIRT1 compared to their respective controls. Hashtags (## p < 0.01, ### p < 0.001) indicate significant mean differences between cells treated with either SRT2104 vs siSIRT1

Fig. 5

SIRT1 reduces anti-apoptotic proteins, BCL-XL and MCL1. A and B SVOG cells were treated with either control media or SRT2104 (50 μmol/L) for 24 h and 48 h. C Cells were transfected with 10 nmol/L of either scrambled siRNA (siNC) or SIRT1 siRNA (siSIRT1) and collected 48 h post transfection. BCL-XL and MCL1 protein levels were determined in cell extracts by western blotting. Cropped images representative of western blot are shown in the upper panels. Densitometric quantifications are relative to cells cultured in respective control media; protein levels were normalized to the abundance of total MAPK (p44/42; loading control). The results are presented as the means ± SEM from 3 independent experiments. Asterisks indicate significant differences from their respective controls (*p < 0.05, **p < 0.01)

Fig. 6

SIRT1 increases cleaved caspase 3 and PARP while decreasing full-length forms of these pro-apoptotic proteins. A-D Cells were treated with either control media or SRT2104 (50 μmol/L) for 24 h and 48 h or with Staurosporine (STS, 50 nmol/L) for 2 h (A-D). F Cells were transfected with 10 nmol/L of either scrambled siRNA (siNC) or SIRT1 siRNA (siSIRT1). At 6 and 24 h post-transfection, the media were changed, and protein was extracted at 48 h post-transfection. Full-length caspase 3 and cleaved caspase 3 protein levels (A and B, respectively), full-length PARP and cleaved PARP (C and D, respectively), were determined in cell extracts by specific antibodies in western blotting; cropped images representative of western blots are presented (E). Densitometric quantifications are relative to cells cultured in control media or transfected with siNC and normalized relative to the abundance of total MAPK (p44/42; loading control). The results are presented as the means ± SEM from 4 independent experiments. Asterisks indicate significant differences from their respective controls (*p < 0.05, ** p < 0.01, ***p < 0.001)

Fig. 7

SIRT1 elevates necroptotic proteins RIPK1 (A,C) and MLKL (B,D). SVOG cells were treated with either control media or SRT2104 (50 μmol/L) for 24 h and 48 h (A and B) or cells were transfected with either scrambled siRNA (siNC) or SIRT1 siRNA (siSIRT1) and the protein was extracted at 48 h post-transfection (C and D). RIPK1 and MLKL protein levels were determined in cell extracts by specific antibodies in western blotting and normalized relative to the abundance of total MAPK (p44/42; loading control). Cropped images representative of western blot are shown in the upper panels. Results are presented as the means ± SEM from 4 independent experiments. Asterisks indicate significant differences from their respective controls (*p < 0.05, **p < 0.01)

Fig. 8

Nec-1 abolishes SRT2104 actions on live and late apoptotic cell populations. SVOG cells were pretreated with Nec-1 (20 μmol/L) for 2 h, followed by incubation for 24 (B) and 48 h (D) with medium only (control) or SRT2104 (50 μmol/L). Then, cells were analyzed using FACS with propidium iodide (PI) and Annexin V staining (representative plots are shown in A-24 h and C-48 h). Results are presented as the means ± SEM from 3 independent experiments. The different letters indicate significant statistical differences at p < 0.05 analyzed using ANOVA followed by Tukey HSD post hoc multiple comparison test

Fig. 9

Nec-1 counteracts SIRT1 actions on pro-apoptotic and necroptotic proteins. SVOG cells were treated with either control media or SRT2104 (50 μmol/L) for 24 h or pretreated with Nec-1 (20 μmol/L) for 2 h. Protein levels (A- cleaved caspase 3, B- cleaved PARP, C-RIPK1 and D-MLKL) were determined in cell extracts by western blotting and normalized relative to the abundance of total MAPK (p44/42; loading control). Cropped image of representative western blots is shown in the upper panel. Results are presented as the means ± SEM from 3 independent experiments. The different letters indicate significant statistical differences at p < 0.05 analyzed using ANOVA followed by Tukey HSD post hoc multiple comparison test

Fig. 10

Illustrative summary depicting diverse actions of SIRT1 in human granulosa cells. SIRT1, activated by SRT2104 was previously shown to elevate cAMP levels [12]. SIRT1 induced genes pivotal for ovulation (EREG and PTGS2) and angiogenesis (FGF2 and VEGFA), most likely in a cAMP-dependent manner. SIRT1 activation and subsequent elevation of cAMP may also promote reduction of anti-apoptotic proteins BCL-XL and MCL1. This triggers the cleavage and activation of caspase 3. Cleaved caspase 3 then cleaves PARP leading consequently to apoptosis. One may therefore suggest, SIRT1-induced cAMP is responsible for the coexistence of luteinization and apoptosis in luteinizing GCs. SIRT1 also activates RIPK1 and MLKL proteins thereby advancing necroptosis. Additionally, activation of RIPK1 can also mediate apoptosis by activating caspase 3. The role of RIPK1 in SIRT1 induced apoptosis and necroptosis is affirmed by the attenuation of apoptotic and necroptotic proteins by Nec-1. Pattern of FACS sorted cells further support the role of SIRT1 as inducer of apoptosis and necroptosis. Ablation of endogenous SIRT1 with siRNA produced opposing actions thereby corroborating the effects observed with SIRT1 activation