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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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10 μM βE2 pretreatment for 0.5 hrs protected primary cultured SD rat retinal cells from apoptosis induced by 100 μM H2O2 treatment for 24 hrs.The PI3K/Akt pathway mediated this process, but the alteration in [Ca2+]i was undetectable. A: The Annexin V/Propidium Iodide staining apoptosis assay; B: Quantitative data of A; C and D: Cell viability and [Ca2+]i quantitative data; 10 μM βE2 pretreatment for 0.5 hrs significantly restored the decrease in cell viability and apoptosis, which was significantly inhibited by 10 μM LY (B, C), but the [Ca2+]i was not significantly altered in all treated groups (D); E: Western blot results, 10 μM βE2 pretreatment for 0.5 hrs promoted p-Akt level, which was inhibited by 10 μM LY pretreatment for 0.5 hrs before βE2 and H2O2 co-treatment. F: Quantitative data of E. Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group by the T-test or one-way ANOVA statistical analysis; ### represents P<0.001 compared with the H2O2 application group by one-way ANOVA statistical analysis; $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B, C, D: n indicates 3 independent replicates with 4 samples per condition per experiment; F: n indicates 3 independent replicates.).
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pone-0077218-g006: 10 μM βE2 pretreatment for 0.5 hrs protected primary cultured SD rat retinal cells from apoptosis induced by 100 μM H2O2 treatment for 24 hrs.The PI3K/Akt pathway mediated this process, but the alteration in [Ca2+]i was undetectable. A: The Annexin V/Propidium Iodide staining apoptosis assay; B: Quantitative data of A; C and D: Cell viability and [Ca2+]i quantitative data; 10 μM βE2 pretreatment for 0.5 hrs significantly restored the decrease in cell viability and apoptosis, which was significantly inhibited by 10 μM LY (B, C), but the [Ca2+]i was not significantly altered in all treated groups (D); E: Western blot results, 10 μM βE2 pretreatment for 0.5 hrs promoted p-Akt level, which was inhibited by 10 μM LY pretreatment for 0.5 hrs before βE2 and H2O2 co-treatment. F: Quantitative data of E. Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group by the T-test or one-way ANOVA statistical analysis; ### represents P<0.001 compared with the H2O2 application group by one-way ANOVA statistical analysis; $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B, C, D: n indicates 3 independent replicates with 4 samples per condition per experiment; F: n indicates 3 independent replicates.).

Mentions: Based on the results of cell viability and apoptosis assay in Figure 1D and H, 100 μM H2O2 treatment for 2 hrs led promoted retinal cell injury but not apoptosis. Therefore, we tested the role of βE2 in anti-apoptosis induced by 100 μM H2O2 for 24 hrs and the inhibitory effect of LY294002. In this experiment, we assayed the cell viability by the MTT assay and apoptosis by Annexin V/Propidium Iodide staining, and meanwhile, [Ca2+]i measurements and Western blotting were performed. The results showed that 10 μM βE2 pretreatment for 0.5 hrs effectively protected the retinal cells from injury and apoptosis induced by 100 μM H2O2-mediated stressing for 24 hrs. Moreover, application of 10 μM LY294002 for 0.5 hrs before βE2 treatment significantly inhibited the βE2-mediated retinal protection against the H2O2-induced cell viability decrease and apoptosis (Figure 6A-C). Nevertheless, the [Ca2+]i showed no alteration in all treated groups compared to the control group (Figure 6D), which further implicated that the βE2-induced increase in the [Ca2+]i is an instantaneous event and that the [Ca2+]i overload induced by H2O2 occurred during the early stage of apoptosis but did not occur at the later stages of apoptosis. Western blot results also showed that 10 μM βE2 pretreatment for 0.5 hrs markedly activated the PI3K/Akt pathway, which was significantly inhibited by 10 μM LY294002 (Figure 6E, F).


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)

10 μM βE2 pretreatment for 0.5 hrs protected primary cultured SD rat retinal cells from apoptosis induced by 100 μM H2O2 treatment for 24 hrs.The PI3K/Akt pathway mediated this process, but the alteration in [Ca2+]i was undetectable. A: The Annexin V/Propidium Iodide staining apoptosis assay; B: Quantitative data of A; C and D: Cell viability and [Ca2+]i quantitative data; 10 μM βE2 pretreatment for 0.5 hrs significantly restored the decrease in cell viability and apoptosis, which was significantly inhibited by 10 μM LY (B, C), but the [Ca2+]i was not significantly altered in all treated groups (D); E: Western blot results, 10 μM βE2 pretreatment for 0.5 hrs promoted p-Akt level, which was inhibited by 10 μM LY pretreatment for 0.5 hrs before βE2 and H2O2 co-treatment. F: Quantitative data of E. Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group by the T-test or one-way ANOVA statistical analysis; ### represents P<0.001 compared with the H2O2 application group by one-way ANOVA statistical analysis; $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B, C, D: n indicates 3 independent replicates with 4 samples per condition per experiment; F: n indicates 3 independent replicates.).
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pone-0077218-g006: 10 μM βE2 pretreatment for 0.5 hrs protected primary cultured SD rat retinal cells from apoptosis induced by 100 μM H2O2 treatment for 24 hrs.The PI3K/Akt pathway mediated this process, but the alteration in [Ca2+]i was undetectable. A: The Annexin V/Propidium Iodide staining apoptosis assay; B: Quantitative data of A; C and D: Cell viability and [Ca2+]i quantitative data; 10 μM βE2 pretreatment for 0.5 hrs significantly restored the decrease in cell viability and apoptosis, which was significantly inhibited by 10 μM LY (B, C), but the [Ca2+]i was not significantly altered in all treated groups (D); E: Western blot results, 10 μM βE2 pretreatment for 0.5 hrs promoted p-Akt level, which was inhibited by 10 μM LY pretreatment for 0.5 hrs before βE2 and H2O2 co-treatment. F: Quantitative data of E. Values shown are the Mean ±SD. *represents P<0.05, **represents P<0.01 and ***represents P<0.001 compared with the control group by the T-test or one-way ANOVA statistical analysis; ### represents P<0.001 compared with the H2O2 application group by one-way ANOVA statistical analysis; $$$ represents P<0.001 compared with the βE2 and H2O2 co-application group by one-way ANOVA statistical analysis. (B, C, D: n indicates 3 independent replicates with 4 samples per condition per experiment; F: n indicates 3 independent replicates.).
Mentions: Based on the results of cell viability and apoptosis assay in Figure 1D and H, 100 μM H2O2 treatment for 2 hrs led promoted retinal cell injury but not apoptosis. Therefore, we tested the role of βE2 in anti-apoptosis induced by 100 μM H2O2 for 24 hrs and the inhibitory effect of LY294002. In this experiment, we assayed the cell viability by the MTT assay and apoptosis by Annexin V/Propidium Iodide staining, and meanwhile, [Ca2+]i measurements and Western blotting were performed. The results showed that 10 μM βE2 pretreatment for 0.5 hrs effectively protected the retinal cells from injury and apoptosis induced by 100 μM H2O2-mediated stressing for 24 hrs. Moreover, application of 10 μM LY294002 for 0.5 hrs before βE2 treatment significantly inhibited the βE2-mediated retinal protection against the H2O2-induced cell viability decrease and apoptosis (Figure 6A-C). Nevertheless, the [Ca2+]i showed no alteration in all treated groups compared to the control group (Figure 6D), which further implicated that the βE2-induced increase in the [Ca2+]i is an instantaneous event and that the [Ca2+]i overload induced by H2O2 occurred during the early stage of apoptosis but did not occur at the later stages of apoptosis. Western blot results also showed that 10 μM βE2 pretreatment for 0.5 hrs markedly activated the PI3K/Akt pathway, which was significantly inhibited by 10 μM LY294002 (Figure 6E, F).

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