Role of protein phosphatases in estrogen-mediated neuroprotection - PubMed (original) (raw)
Role of protein phosphatases in estrogen-mediated neuroprotection
Kun Don Yi et al. J Neurosci. 2005.
Abstract
The signaling pathways that mediate neurodegeneration are complex and involve a balance between phosphorylation and dephosphorylation of signaling and structural proteins. We have shown previously that 17beta-estradiol and its analogs are potent neuroprotectants. The purpose of this study was to delineate the role of protein phosphatases (PPs) in estrogen neuroprotection against oxidative stress and excitotoxicity. HT-22 cells, C6-glioma cells, and primary rat cortical neurons were exposed to the nonspecific serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A at various concentrations in the presence or absence of 17beta-estradiol and/or glutamate. Okadaic acid and calyculin A caused a dose-dependent decrease in cell viability in HT-22, C6-glioma, and primary rat cortical neurons. 17beta-Estradiol did not show protection against neurotoxic concentrations of either okadaic acid or calyculin A in these cells. In the absence of these serine/threonine protein phosphatase inhibitors, 17beta-estradiol attenuated glutamate toxicity. However, in the presence of effective concentrations of these protein phosphatase inhibitors, 17beta-estradiol protection against glutamate toxicity was lost. Furthermore, glutamate treatment in HT-22 cells and primary rat cortical neurons caused a 50% decrease in levels of PP1, PP2A, and PP2B protein, whereas coadministration of 17beta-estradiol with glutamate prevented the decrease in PP1, PP2A, and PP2B levels. These results suggest that 17beta-estradiol may protect cells against glutamate-induced oxidative stress and excitotoxicity by activating a combination of protein phosphatases.
Figures
Figure 1.
Effects of okadaic acid on estrogen-mediated neuroprotection from glutamate neurotoxicity. HT-22 cells (passages 18-25), C6-glioma cells (passages 40-49), or primary rat cortical neurons were seeded into 96-well plates at a density of 3500 or 25,000 cells per well. A, HT-22 cells treated simultaneously with 100 n
m
okadaic acid, 10 m
m
glutamate, and/or 10 μ
m
17β-estradiol. B, C6-glioma cells treated simultaneously with 100 n
m
okadaic acid, 20 m
m
glutamate, and/or 10 μ
m
17β-estradiol. C, Primary cortical neurons treated simultaneously with 50 n
m
okadaic acid, 50 μ
m
glutamate, and/or 100 n
m
17β-estradiol. Cell viability was determined by calcein AM assay after 24 h exposure to the various compounds. All data were normalized to percentage survival of vehicle control. Data are represented as mean ± SEM for n = 6. *p < 0.05 versus vehicle control; †p < 0.05 versus glutamate-treated group.
Figure 2.
Dose-dependent neurotoxicity of okadaic acid (A) and calyculin A (B). HT-22 cells (passages 18-25) and C6-glioma cells (passages 40-49) were seeded into 96-well plates at a density of 3500 cells per well, and primary rat cortical neurons were seeded into 96-well plates at a density of 25,000 cells per well. Okadaic acid or calyculin A were added at varying concentrations. Cell viability was determined by calcein AM assay after 24 h exposure to the various protein phosphatase inhibitors. All data were normalized to percentage survival of vehicle control. Data are represented as mean ± SEM for n = 6. *p < 0.05 versus vehicle control.
Figure 3.
Effects of 17β-estradiol on okadaic acid- or calyculin A-induced neurotoxicity. HT-22 cells (passages 18-25) and C6-glioma cells (passages 40-49) were seeded into 96-well plates at a density of 3500 cells per well, and primary rat cortical neurons were seeded into 96-well plates at a density of 25,000 cells per well. HT-22 cells treated with 100 n
m
okadaic acid or 1 n
m
calyculin A and simultaneous (A) or 2 h pretreatment (B) of 17β-estradiol (E2) at varying concentrations are shown. C6-glioma cells treated with 100 n
m
okadaic acid or 500 p
m
calyculin A and simultaneous (C) or 2 h pretreatment (D) of 17β-estradiol at varying concentrations are shown. Primary cortical neurons treated with 50 n
m
okadaic acid or 10 n
m
calyculin A and simultaneous (E) or 2 h pretreatment (F) of 17β-estradiol at varying concentrations are shown. Cell viability was determined by calcein AM assay after 24 h exposure to the various protein phosphatase inhibitors and 17β-estradiol. All data were normalized to percentage survival of vehicle control. Data are represented as mean ± SEM for n = 6. *p < 0.05 versus vehicle control.
Figure 4.
Time course of the effects 17β-estradiol (B), glutamate (A), and their combination (C) on PP2A protein levels. HT-22 cells were treated with 10 m
m
glutamate and/or 10 μ
m
17β-estradiol. Cells were harvested at the times indicated for Western blot analysis of PP2A. The graphs represent relative OD as a percentage of time 0 control. Data are represented as mean ± SEM for n = 3. *p < 0.05 versus time 0 control.
Figure 5.
PP1 (A), PP2A (B), and PP2B (C) protein levels in response to 17β-estradiol in the presence and absence glutamate and/or okadaic acid in HT-22 cells. HT-22 cells were treated with 100 n
m
okadaic acid (OA), 10 m
m
glutamate (Glut), and/or 10 μ
m
17β-estradiol (E2). Cells were harvested after 24 h of treatment for Western blot analysis of PP1, PP2A, and PP2B. Data are represented as mean ± SEM for n = 3. *p < 0.05 versus control; †p < 0.05 versus glutamate-treated group.
Figure 6.
PP1 (A), PP2A (B), and PP2B (C) protein levels in response to 17β-estradiol in the presence and absence of 50 μ
m
glutamate and/or 50 n
m
okadaic acid in primary cortical neurons. Primary rat cortical neurons were treated with 50 n
m
okadaic acid (OA), 50 μ
m
glutamate (Glut), and/or 100 n
m
17β-estradiol (E2). Cells were harvested after 24 h of treatment for Western blot analysis of PP1, PP2A, and PP2B. Data are represented as mean ± SEM for n = 3. *p < 0.05 versus control; †p < 0.05 versus glutamate-treated group.
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