Krüppel-like factor 12 regulates aging ovarian granulosa cell apoptosis by repressing SPHK1 transcription and sphingosine-1-phosphate (S1P) production

Figure 1. Increased expression of both reactive oxygen species (ROS) and KLF12 in ovarian GCs of aging patients.A, expression levels of ROS in ovarian GCs of young and old patients. The green color represents the levels of ROS activity. B, S1P expression concentrations in ovarian GCs of young and old patients. The results of each replicate experiment are marked as circles and squares. C and D, mRNA expression of SPHK1/KLF12 in ovarian GCs of young and old patients. E, correlation analysis of mRNA expression of SPHK1 and KLF12 in human ovarian GCs. The analytical result shows a negative correlation (correlation coefficient r = −0.4837) between SPHK1 and KLF12. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. GC, granulosa cell; KLF12, Krüppel-like factor 12; S1P, sphingosine-1-phosphate; SPHK1, sphingosine kinase 1.

Figure 2. Dynamic changes of KLF12 in H2O2-induced oxidative stress model in KGN cells.A and B, changes in mRNA expression of KLF12/SPHK1 in KGN cells stimulated by different concentrations (0, 50, 100, and 200 μM) of H2O2. C, changes in protein expression of KLF12/SPHK1 in KGN cells stimulated by different concentrations of H2O2. Protein expression was normalized to actin levels. D and E, changes in mRNA expression of KLF12/SPHK1 in KGN cells under different durations (0, 12, 24, and 48 h) of H2O2 stimulation. F, protein expression changes of KLF12/SPHK1 in KGN cells under different durations of H2O2 stimulation. Protein expression was normalized to actin levels. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. H2O2, hydrogen peroxide; KLF12, Krüppel-like factor 12; SPHK1, sphingosine kinase 1.

Figure 3. Knockdown of KLF12 delays H2O2-induced apoptosis in KGN cells.A, apoptosis levels in KGN cells with different concentrations of KLF12 overexpression. Adenovirus ad-FLAG-KLF12 was used at 0, 10, and 20 MOI, with corresponding Ad-LacZ at 20, 10, and 0 MOI. B, expression levels of KLF12 in KGN cells with different concentrations of KLF12 overexpression. C, detection of apoptosis-related factors in KGN cells with different concentrations of KLF12 overexpression. Caspase-3 was used to detect cell apoptosis. Cleaved caspase-3 was used to assess the degree of cell apoptosis. D, flow cytometry analysis of apoptosis in KGN cells with different concentrations of KLF12 overexpression. From left to right: Ad-LacZ, ad-FLAG-KLF12 (10 MOI), and ad-FLAG-KLF12 (20 MOI). PI was added for cell analysis. E, knockdown of KLF12 delays H2O2-induced apoptosis in KGN cells. F, knockdown of KLF12 delays H2O2-induced apoptosis-related factor expression in KGN cells. G, flow cytometry analysis of the effect of KLF12 knockdown on H2O2-induced apoptosis in KGN cells. Four groups were set up: NC (no treatment), siKLF12, NC + H2O2, and siKLF12 + H2O2. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. H2O2, hydrogen…

Figure 4. KLF12 transcriptionally represses SPHK1 expression and reduces S1P production.A and B, SPHK1 mRNA/protein expression decreases with increasing KLF12 expression. Adenovirus ad-FLAG-KLF12 was applied at 0, 10, and 20 MOI, whereas the corresponding Ad-LacZ was applied at 20, 10, and 0 MOI. C, S1P expression concentration decreases with increasing KLF12 expression. D, sequence design diagram of SPHK1 promoter (Luc1/Luc2/Luc3). The sequence positions for SPHK1-Luc1 are −4979 to −4576, for SPHK1-Luc2 are −6777 to −6328, and for SPHK1-Luc3 are −6777 to −4576. The red-highlighted sequence CAGTGGG is the predicted binding site for KLF12 with the SPHK1 promoter. E–G, luciferase reporter assay of SPHK1 promoter (Luc1/Luc2/Luc3). When FLAG-KLF12 is expressed in the KGN cell line, SPHK1 luciferase activity significantly decreases by more than half. H, chromatin immunoprecipitation (ChIP) assay of SPHK1 promoter (Luc1/Luc2) and KLF12. KLF12 displays strong binding efficiency with the SPHK1 promoter (Luc1/Luc2). I, avidin–biotin conjugate DNA (ABCD) precipitation assay for SPHK1 of WT and MUT. KGN cells transfected with Ad-FLAG-KLF12 were lysed, and the binding ability between DNA lysate…

Figure 5. The overexpression of SPHK1 partially rescues H2O2or KLF12-induced apoptosis in KGN and mGC.A–C, the expression levels of various apoptotic markers were evaluated to unravel the underlying mechanism. Treatment with H2O2 or KLF12 increased cleaved caspase-3 expression levels, indicating augmented apoptosis. However, the overexpression of SPHK1 significantly attenuated the apoptosis levels (with increased expression levels of p-FOXO1 and p-AKT1). D, for cell flow cytometry analysis, propidium iodide (PI) was added, and different types of apoptotic cells were distinguished based on their Annexin V and PI labeling. E–G, the overexpression of SPHK1 partly rescued KLF12-induced apoptosis in KGN/mGC cells, which was associated with increased expression levels of p-FOXO1 and p-AKT1. H, a cell flow cytometry plot was used to evaluate KLF12 rescue of H2O2-induced apoptosis in KGN cells. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. H2O2, hydrogen peroxide; KLF12, Krüppel-like factor 12; mGC, mouse granulosa cell; SPHK1, sphingosine kinase 1.

Figure 6. SPHK1 supplementation partially rescues 3-NP/KLF12-induced apoptosis in ovarian GCs in an ovarian culture model.A, SPHK1 supplementation mitigates 3-NP-induced apoptosis-related factors as analyzed through protein assays in an ovarian culture model. B–D, p-FOXO1, p-AKT1, and cleaved caspase-3 expression levels indicate changes in the 3-NP-induced ovarian culture model. 3-NP treatment increases apoptosis in ovarian GCs (elevated cleaved caspase-3 expression), but SPHK1 expression significantly counteracts the increase in apoptosis levels (increased p-FOXO1 and p-AKT1 expression levels). E, SPHK1 supplementation mitigates KLF12-induced apoptosis-related factors as detected through protein assays in an ovarian culture model. F–H, p-FOXO1, p-AKT1, and cleaved caspase-3 expression levels indicate changes in the KLF12-induced ovarian culture model. Overexpression of KLF12 increases apoptosis in ovarian GCs (elevated cleaved caspase-3 expression), but SPHK1 expression significantly counteracts the increase in apoptosis levels (increased p-FOXO1 and p-AKT1 expression levels). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. 3-NP, 3-nitropropionic acid; GC, granulosa cell; KLF12, Krüppel-like…

Figure 7. KLF12 and SPHK1 exhibit negative correlation in 3-NP-induced in vivo mouse ovaries.A and B, mRNA expression of KLF12 and SPHK1 in 3-NP-induced in vivo mouse ovaries. C–F, protein expression of KLF12, SPHK1, and cleaved caspase-3 in 3-NP-induced in vivo mouse ovaries. In 3-NP-induced mouse ovaries, apoptotic factor (cleaved caspase-3) expression increases and anti-apoptotic factors (p-FOXO1 and p-AKT1) expression decreases. KLF12 expression significantly increases, and SPHK1 expression significantly decreases, presenting a negative correlation. G, KLF12 and SPHK1 show a negative correlation in 3-NP-induced in vivo mouse ovaries. H, immunohistochemistry reveals increased expression of cleaved caspase-3 and KLF12 in 3-NP-induced oxidative stress ovaries. The scale bar represents 50 μm. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. 3-NP, 3-nitropropionic acid; KLF12, Krüppel-like factor 12; SPHK1, sphingosine kinase 1.

Supporting Figure S1. High expression of Ad-KLF12 DN does not affect the apoptosis of KGN cells.A, investigating the effect of Ad-KLF12 DN, a high-expression adenovirus of KLF12 with functional structure deletion, on apoptosis. B and C, assessing the impact of high expression of Ad-KLF12 DN on the protein expression of SPHK1 and S1P production, which was found to be non-significant. The overexpression of Ad-KLF12 DN in KGN cells did not affect apoptosis, thus ruling out any non-specific effects of KLF12 overexpression. Statistical significance: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Supporting Figure S2. High expression of Ad-SPHK1 DN cannot rescue apoptosis of KGN cells.A, investigating the effect of high expression of Ad-SPHK1 DN on S1P concentration. B and C, examining the impact of high expression of Ad-SPHK1 DN on H2O2/KLF12-induced apoptosis, which was found to be non-rescuable. D and E, analyzing changes in apoptotic factors in H2O2/KLF12-induced KGN cells by high expression of Ad-SPHK1 DN. The overexpression of Ad-SPHK1 DN in KGN cells did not affect apoptosis, thus ruling out any non-specific effects of SPHK1 overexpression. Ad-SPHK1 DN is a high-expression adenovirus of SPHK1 with functional structure deletion. Statistical significance: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.