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The involvement of PI3K-mediated and L-VGCC-gated transient Ca2+ influx in 17β-estradiol-mediated protection of retinal cells from H2O2-induced apoptosis with Ca2+ overload.

Feng Y, Wang B, Du F, Li H, Wang S, Hu C, Zhu C, Yu X - PLoS ONE (2013)

Bottom Line: Fluo-3AM was used as a Ca(2+) indicator to detect [Ca(2+)]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining.Results showed: a) 100 μM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca(2+)]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca(2+)]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca(2+)]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca(2+) stores; d) importantly, the transiently increased [Ca(2+)]i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca(2+) channels (L-VGCC), but the increased [Ca(2+)]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca(2+)]i increase through L-VGCC and PI3K pathway.These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

ABSTRACT
Intracellular calcium concentration ([Ca(2+)]i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca(2+)]i and its control mechanisms in process of hydrogen peroxide (H2O2)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca(2+) indicator to detect [Ca(2+)]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca(2+)]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca(2+)]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca(2+)]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca(2+) stores; d) importantly, the transiently increased [Ca(2+)]i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca(2+) channels (L-VGCC), but the increased [Ca(2+)]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca(2+)]i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.

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The effect of the PI3K inhibitor LY294002 (LY) on the cell viability and the [Ca2+]i of primary cultured SD rat retinal cells in H2O2 injury and βE2 protection.A: Western blot results of the activation of the PI3K/Akt pathway after βE2 treatment for 0.5 hrs; B: Quantitative data of A; C, E, G, and I: Cell viability quantitative data; D, F, H, and J: [Ca2+]i quantitative data; C and D: The effects of LY treatments for 24 hrs and 0.5 hrs on the cell viability and the resting [Ca2+]i; E and F: The inhibitory effect of LY pretreatment for 0.5 hrs on the increased cell viability and [Ca2+]i induced by 10 μM βE2 treatment for 0.5 hrs (10 μM LY in E, 10 μM and 20 μM LY in F); G and H: The effect of LY pretreatment for 0.5 hrs on the decreased cell viability and increased [Ca2+]i induced by 100 μM H2O2 treatment for 2 hrs (10 μM LY in G, 10 μM and 20 μM LY in H); I and J: The dose-dependent attenuating impact of 20-50 μM LY pretreatment for 0.5 hrs on the βE2 retinal protective role against H2O2 injury, which is associated with the dose-dependent attenuation of the increased [Ca2+]i (Protocol of drug application: LY for 0.5 hrs, E2 for 0.5 hrs and H2O2 for 2 hrs). Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group; # represents P<0.05, ## represents P<0.01 and ### represents P<0.001 compared with the βE2 (E, F) or H2O2 (G, I, J) application groups; $ represents P<0.05, $$ represents P<0.01 and $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B: n indicates 3 independent replicates; C, E, G, I: n indicates 3 independent replicates with 4 samples per condition per experiment; D, F, H, J: n indicates 3 independent replicates with 5 samples per condition per experiment.).
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pone-0077218-g005: The effect of the PI3K inhibitor LY294002 (LY) on the cell viability and the [Ca2+]i of primary cultured SD rat retinal cells in H2O2 injury and βE2 protection.A: Western blot results of the activation of the PI3K/Akt pathway after βE2 treatment for 0.5 hrs; B: Quantitative data of A; C, E, G, and I: Cell viability quantitative data; D, F, H, and J: [Ca2+]i quantitative data; C and D: The effects of LY treatments for 24 hrs and 0.5 hrs on the cell viability and the resting [Ca2+]i; E and F: The inhibitory effect of LY pretreatment for 0.5 hrs on the increased cell viability and [Ca2+]i induced by 10 μM βE2 treatment for 0.5 hrs (10 μM LY in E, 10 μM and 20 μM LY in F); G and H: The effect of LY pretreatment for 0.5 hrs on the decreased cell viability and increased [Ca2+]i induced by 100 μM H2O2 treatment for 2 hrs (10 μM LY in G, 10 μM and 20 μM LY in H); I and J: The dose-dependent attenuating impact of 20-50 μM LY pretreatment for 0.5 hrs on the βE2 retinal protective role against H2O2 injury, which is associated with the dose-dependent attenuation of the increased [Ca2+]i (Protocol of drug application: LY for 0.5 hrs, E2 for 0.5 hrs and H2O2 for 2 hrs). Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group; # represents P<0.05, ## represents P<0.01 and ### represents P<0.001 compared with the βE2 (E, F) or H2O2 (G, I, J) application groups; $ represents P<0.05, $$ represents P<0.01 and $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B: n indicates 3 independent replicates; C, E, G, I: n indicates 3 independent replicates with 4 samples per condition per experiment; D, F, H, J: n indicates 3 independent replicates with 5 samples per condition per experiment.).

Mentions: βE2 plays a protective role in the retina via the PI3K/Akt pathway [28]. Our results showed that βE2 protected primary cultured SD rat retinal cells from H2O2 injury, which was associated with a transient [Ca2+]i increase (Figure 2). Therefore, we hypothesized that βE2 plays a protective role in our study model by activating the PI3K pathway and then transiently increasing [Ca2+]i. To test this hypothesis, we performed the following experiments using the PI3K inhibitor LY294002. First, we confirmed that 10 μM βE2 treatment for 0.5 hrs up-regulated the p-Akt level via Western blotting (Figure 5A, B). Second, we measured the effects of LY294002 on the cell viability and the [Ca2+]i of the retinal cells and found that 1-50 μM LY294002 treatment for 24 hrs dose-dependently decreased the cell viability (Figure 5C), but treatment for 0.5 hrs had no effect on the resting [Ca2+]i (Figure 5D). Third, we detected the inhibitory effects of LY294002 on the alteration of [Ca2+]i and cell viability due to 10 μM βE2 treatment for 0.5 hrs or 100 μM H2O2 treatment for 2 hrs. Results showed that pretreatment for 0.5 hrs with 10 μM or 20 μM LY294002 significantly attenuated the increased cell viability and [Ca2+]i due to βE2 (Figure 5E, F). However, 10 μM LY294002 did not reverse the cell viability decrease induced by H2O2 but instead promoted the decrease in cell viability (Figure 5G). In addition, both 10 μM and 20 μM LY294002 had no effect on the [Ca2+]i increase induced by H2O2 (Figure 5H). PI3K was involved in the βE2-induced increase of [Ca2+]i and cell viability but was not involved in the H2O2-induced [Ca2+]i increase and cell viability decrease. Fourth, we verified that PI3K-mediated βE2 protection against H2O2 injury was associated with transiently up-regulating [Ca2+]i. As shown in Figures 5I and J, 20-50 μM LY294002 dose-dependently attenuated the βE2-mediated protective effect against H2O2 injury and dose-dependently restored the increased [Ca2+]i induced by co-treatment with 10 μM βE2 for 0.5 hrs and 100 μM H2O2 for 2 hrs.


The involvement of PI3K-mediated and L-VGCC-gated transient Ca2+ influx in 17β-estradiol-mediated protection of retinal cells from H2O2-induced apoptosis with Ca2+ overload.

Feng Y, Wang B, Du F, Li H, Wang S, Hu C, Zhu C, Yu X - PLoS ONE (2013)

The effect of the PI3K inhibitor LY294002 (LY) on the cell viability and the [Ca2+]i of primary cultured SD rat retinal cells in H2O2 injury and βE2 protection.A: Western blot results of the activation of the PI3K/Akt pathway after βE2 treatment for 0.5 hrs; B: Quantitative data of A; C, E, G, and I: Cell viability quantitative data; D, F, H, and J: [Ca2+]i quantitative data; C and D: The effects of LY treatments for 24 hrs and 0.5 hrs on the cell viability and the resting [Ca2+]i; E and F: The inhibitory effect of LY pretreatment for 0.5 hrs on the increased cell viability and [Ca2+]i induced by 10 μM βE2 treatment for 0.5 hrs (10 μM LY in E, 10 μM and 20 μM LY in F); G and H: The effect of LY pretreatment for 0.5 hrs on the decreased cell viability and increased [Ca2+]i induced by 100 μM H2O2 treatment for 2 hrs (10 μM LY in G, 10 μM and 20 μM LY in H); I and J: The dose-dependent attenuating impact of 20-50 μM LY pretreatment for 0.5 hrs on the βE2 retinal protective role against H2O2 injury, which is associated with the dose-dependent attenuation of the increased [Ca2+]i (Protocol of drug application: LY for 0.5 hrs, E2 for 0.5 hrs and H2O2 for 2 hrs). Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group; # represents P<0.05, ## represents P<0.01 and ### represents P<0.001 compared with the βE2 (E, F) or H2O2 (G, I, J) application groups; $ represents P<0.05, $$ represents P<0.01 and $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B: n indicates 3 independent replicates; C, E, G, I: n indicates 3 independent replicates with 4 samples per condition per experiment; D, F, H, J: n indicates 3 independent replicates with 5 samples per condition per experiment.).
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pone-0077218-g005: The effect of the PI3K inhibitor LY294002 (LY) on the cell viability and the [Ca2+]i of primary cultured SD rat retinal cells in H2O2 injury and βE2 protection.A: Western blot results of the activation of the PI3K/Akt pathway after βE2 treatment for 0.5 hrs; B: Quantitative data of A; C, E, G, and I: Cell viability quantitative data; D, F, H, and J: [Ca2+]i quantitative data; C and D: The effects of LY treatments for 24 hrs and 0.5 hrs on the cell viability and the resting [Ca2+]i; E and F: The inhibitory effect of LY pretreatment for 0.5 hrs on the increased cell viability and [Ca2+]i induced by 10 μM βE2 treatment for 0.5 hrs (10 μM LY in E, 10 μM and 20 μM LY in F); G and H: The effect of LY pretreatment for 0.5 hrs on the decreased cell viability and increased [Ca2+]i induced by 100 μM H2O2 treatment for 2 hrs (10 μM LY in G, 10 μM and 20 μM LY in H); I and J: The dose-dependent attenuating impact of 20-50 μM LY pretreatment for 0.5 hrs on the βE2 retinal protective role against H2O2 injury, which is associated with the dose-dependent attenuation of the increased [Ca2+]i (Protocol of drug application: LY for 0.5 hrs, E2 for 0.5 hrs and H2O2 for 2 hrs). Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group; # represents P<0.05, ## represents P<0.01 and ### represents P<0.001 compared with the βE2 (E, F) or H2O2 (G, I, J) application groups; $ represents P<0.05, $$ represents P<0.01 and $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B: n indicates 3 independent replicates; C, E, G, I: n indicates 3 independent replicates with 4 samples per condition per experiment; D, F, H, J: n indicates 3 independent replicates with 5 samples per condition per experiment.).
Mentions: βE2 plays a protective role in the retina via the PI3K/Akt pathway [28]. Our results showed that βE2 protected primary cultured SD rat retinal cells from H2O2 injury, which was associated with a transient [Ca2+]i increase (Figure 2). Therefore, we hypothesized that βE2 plays a protective role in our study model by activating the PI3K pathway and then transiently increasing [Ca2+]i. To test this hypothesis, we performed the following experiments using the PI3K inhibitor LY294002. First, we confirmed that 10 μM βE2 treatment for 0.5 hrs up-regulated the p-Akt level via Western blotting (Figure 5A, B). Second, we measured the effects of LY294002 on the cell viability and the [Ca2+]i of the retinal cells and found that 1-50 μM LY294002 treatment for 24 hrs dose-dependently decreased the cell viability (Figure 5C), but treatment for 0.5 hrs had no effect on the resting [Ca2+]i (Figure 5D). Third, we detected the inhibitory effects of LY294002 on the alteration of [Ca2+]i and cell viability due to 10 μM βE2 treatment for 0.5 hrs or 100 μM H2O2 treatment for 2 hrs. Results showed that pretreatment for 0.5 hrs with 10 μM or 20 μM LY294002 significantly attenuated the increased cell viability and [Ca2+]i due to βE2 (Figure 5E, F). However, 10 μM LY294002 did not reverse the cell viability decrease induced by H2O2 but instead promoted the decrease in cell viability (Figure 5G). In addition, both 10 μM and 20 μM LY294002 had no effect on the [Ca2+]i increase induced by H2O2 (Figure 5H). PI3K was involved in the βE2-induced increase of [Ca2+]i and cell viability but was not involved in the H2O2-induced [Ca2+]i increase and cell viability decrease. Fourth, we verified that PI3K-mediated βE2 protection against H2O2 injury was associated with transiently up-regulating [Ca2+]i. As shown in Figures 5I and J, 20-50 μM LY294002 dose-dependently attenuated the βE2-mediated protective effect against H2O2 injury and dose-dependently restored the increased [Ca2+]i induced by co-treatment with 10 μM βE2 for 0.5 hrs and 100 μM H2O2 for 2 hrs.

Bottom Line: Fluo-3AM was used as a Ca(2+) indicator to detect [Ca(2+)]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining.Results showed: a) 100 μM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca(2+)]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca(2+)]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca(2+)]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca(2+) stores; d) importantly, the transiently increased [Ca(2+)]i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca(2+) channels (L-VGCC), but the increased [Ca(2+)]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca(2+)]i increase through L-VGCC and PI3K pathway.These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

ABSTRACT
Intracellular calcium concentration ([Ca(2+)]i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca(2+)]i and its control mechanisms in process of hydrogen peroxide (H2O2)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca(2+) indicator to detect [Ca(2+)]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca(2+)]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca(2+)]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca(2+)]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca(2+) stores; d) importantly, the transiently increased [Ca(2+)]i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca(2+) channels (L-VGCC), but the increased [Ca(2+)]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca(2+)]i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.

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Related in: MedlinePlus