GSTT1 is upregulated by oxidative stress through p38-MK2 signaling pathway in human granulosa cells: possible association with mitochondrial activity

Abstract

We previously reported that GSTT1 was upregulated in human granulosa cells during aging and that activation and localization of p38 MAPK was changed in parallel. Although oxidative stress is responsible for these changes, the age-associated expression of GSTT1 regulated by MAPKs and the role of GSTT1 in aged granulosa cells remain unclear. Therefore, we examined the relationship between the expression of GSTT1 and MAPK signaling pathways using human granulosa-like KGN cells stimulated with H(2)O(2) in the presence or absence of various MAPK inhibitors. Interestingly, H(2)O(2)-induced GSTT1 was only inhibited by a p38 inhibitor. An inhibitor of MK2, a downstream regulator of p38, also diminished H(2)O(2)-induced GSTT1 upregulation. Notably, both p38 and MK2 were significantly inactivated in cells carrying an shRNA construct of GSTT1 (∆GSTT1 cells), suggesting that the p38-MK2 pathway is essential for age-associated upregulation of GSTT1. The relevance of GSTT1 in mitochondrial activity was then determined. ∆GSTT1 cells displayed enhanced polarization of mitochondrial membrane potential without increasing the apoptosis, suggesting that the age-associated upregulation of GSTT1 may influence the mitochondrial activity of granulosa cells.

Figure 1

Effects of MAPK inhibitors on the expression of GSTT1 in KGN cells stimulated with H2O2. Cells were treated with H₂O₂ at 200 μM in the presence or absence of SB203580, SP600125 or PD98059 at 10 μM for 24 h and then subjected to the immunofluorescence analysis (A). The primary antibody against GSTT1 was probed with anti-rabbit IgG-Alexa488 (Green). Cells were counterstained with Hoechst 33342 at 10 μM (Blue). Magnification: ×200. A bar graph represent the mean fluorescence intensity per cell ± SEM (B, C). One-way ANOVA: (B) P < 0.05; (C) P < 0.01.

Figure 2

Effects of the MK2 inhibitor on the expression of GSTT1 in KGN cells stimulated with H₂O₂ Cells were treated with H₂O₂ at 200 μM with or without SB203580 at 10 μM or CMPD1 at 330 nM for 24 h and subjected to immunofluorescence analysis (A). The primary antibody against GSTT1 was probed with anti-rabbit IgG-Alexa488 (Green). Cells were counterstained with Hoechst 33342 at 10 μM (Blue). Magnification: ×200. A bar graph represent the mean fluorescence intensity per cell ± SEM (B, C). One-way ANOVA: (B) P < 0.05; (C) P < 0.01.

Figure 3

Depletion of GSTT1 inactivates the p38–MK2 signaling pathway. KGN cells (wild–type, ΔLamin ΔGSTT1 or Δp38α cells) were treated with or without H₂O₂ at 200 μM for 24 h before investigation of the expression of GSTT1 and activation of p38 and MK2 by immunofluorescence analysis (A). The primary antibodies against GSTT1, phosphorylated p38 and phosphorylated MK2 were probed with anti-rabbit IgG–Alexa488 (Green). Cells were counterstained with Hoechst 33342 at 10 μM (Blue). Magnification: ×200. Bar graphs represent the mean fluorescence intensity per cell ± SEM (B). One–way ANOVA: (B, GSTT1) P < 0.001, (B, p-p38) P < 0.05, (B, p-MK2) P < 0.001.

Figure 4

Depletion of GSTT1 prevents the cytoplasmic activation of p38. Cells stimulated with or without H₂O₂ were subjected to fractionation of cytosolic and nuclear proteins. The activity of p38 in each fraction was then analyzed by immunoblotting (A: cytoplasm, B: nuclei). Fifteen micrograms of total protein were used for each lane. Bar graphs represent the mean band intensity ± SEM. (A) Student's t–test: P < 0.05.

Figure 5

Depletion of GSTT1 enhances mitochondrial activity. (A) Wild-type, ΔLamin or ΔGSTT1 cells were stimulated with H₂O₂, and the mitochondrial membrane potential of each cell type was observed by staining with Mitotracker CMXRos at 100 nM. A bar graph represent the fold increase in mean fluorescence intensity ± SEM. One–way ANOVA: P < 0.001. (B) Frequency of apoptosis before and after treatment with H₂O₂ was measured by TUNEL assay. The number of apoptotic cells was counted and divided by the total number of cells per field. A bar graph represent the frequency of apoptosis ± SEM.