Estrogen and tamoxifen reverse manganese-induced glutamate transporter impairment in astrocytes - PubMed (original) (raw)

Comparative Study

Estrogen and tamoxifen reverse manganese-induced glutamate transporter impairment in astrocytes

Eun-Sook Y Lee et al. J Neurochem. 2009 Jul.

Abstract

Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson's disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17beta-Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn-induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn-induced glutamate uptake inhibition, primarily via the up-regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn-induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor beta1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor beta1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn-induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.

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Figures

Fig. 1

Fig. 1

E2/TX protects astrocytes from Mn toxicity in astroglial cultures. (a) Both E2 receptor subtypes, ER-α and ER-β, are expressed in astrocytic cultures. (b) Mn induced astrocytic cytotoxicity occurs in a time- and concentration-dependent manner. Cell viability was assessed by MTT assay as described in the Methods section. (c) E2/TX protects astrocytes from Mn toxicity. Astrocytes were pre-treated for 24 h with E2 (5 and 10 nM) or with TX (0.5 and 1 μM) prior to the addition of Mn (500 μM), followed by the MTT assay. (###p < 0.001 vs. control; **p < 0.01, ***p < 0.001 vs. the Mn treatment; Tukey's test following

anova

). Data are expressed as the mean ± SEM (n = 5–8). Experiments were performed in three independent sets of cultures.

Fig. 2

Fig. 2

E2/TX reverses Mn-induced glutamate uptake inhibition in astrocytes. After treatment with Mn or E2/TX, cells were incubated with 100 nM of unlabeled glutamate containing 0.25 μCi of [3H]glutamate (specific activity: 49.0 Ci/mmol) for 10 min. The uptake was terminated by washing the cells with ice-cold PBS buffer as described in the Methods section. (a) Glutamate uptake was THA-sensitive, Na+-dependent and GLAST subtype-sensitive. [3H]glutamate uptake was measured in uptake buffer, Na+-free medium, in the presence of THA (a non-selective inhibitor of glutamate transporters), MSO (an inhibitor of glutamine synthase) or DHK (a selective inhibitor of GLT-1). (MSO, methionine sulfoximine; THA,

dl

-threo-μ-hydroxyaspartic acid; DHK, dihydrokainic acid). (b) Mn (6 h) inhibited glutamate uptake in a concentration-dependent manner. (c) E2/TX (24 h) increased glutamate uptake. (d) E2/TX (18 h pre-treatment) reversed Mn (6 h)-induced glutamate uptake inhibition. (e) E2/TX-induced enhancement of glutamate uptake was ER-dependent. ###p < 0.001 vs. control (d); *p < 0.05, **p < 0.01, ***p < 0.001, ##p < 0.01 vs. control (b,c,e) or Mn treatment (d); Tukey's test following

anova

. Data are expressed as the mean ± SEM (n = 5–8).

Fig. 3

Fig. 3

Mn leads to glutamate accumulation in the astrocytic media, whereas E2/TX reverses this effect. Cells were treated for 6 or 24 h with Mn or E2/TX, after which 20 μM glutamate were added to the cell cultures, followed by 30 min of incubation. Glutamate levels in the media were measured by a fluorimetric method. (a) Astrocytes were treated for 6 h with Mn. (b) Astrocytes were treated for 24 h with Mn or E2/TX. For one set of each experiment, cells were pre-treated for 24 h with E2 20 nM prior to Mn (250 μM for 6 or 24 h) treatment. a_p_ < 0.05 vs. control, b_p_ < 0.05 vs. Mn 250 μM, *p < 0.05, **p < 0.01 vs. control; Tukey's test following

anova

. Data are expressed as the mean ± SEM (n = 5–8).

Fig. 4

Fig. 4

Mn-induced inhibition of GLAST protein expression in whole cell lysates is reversed by E2/TX. (a) Treatment of astrocytes for 6 h with Mn decreased GLAST expression in a concentration-dependent manner. (b) Treatment of astrocytes for 24 h with E2 (20 nM) and TX (1 and 2 μM) increased GLAST protein expression. (c) Pre-treatment of E2 (10 and 20 nM) and TX (1 μM) for 18 h prior to Mn treatment for 6 h attenuated the Mn-induced inhibitory effects of GLAST expression. Western blot data were also quantified. ##p < 0.01, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; Tukey's test following

anova

. Data are expressed as the mean ± SEM (n = 3). (d) Immunocytochemistry study showed that E2 and TX increased astrocytic GLAST expression after 24 h, whereas Mn inhibited GLAST protein expression after 6 h.

Fig. 5

Fig. 5

E2/TX enhances GLAST expression on the astroglial plasma membranes and reverses Mn-induced reduction by promoting GLAST trafficking. Astroglial plasma membranes were isolated by biotinylation of surface membrane proteins using a biotinylation kit (Pierce) followed by western blot analysis. All samples were adjusted to contain the same amount of proteins prior to loading on the gel. Mn (500 μM) reduced, while E2/TX increased GLAST expression on astroglial cell surface membranes. E2 reversed the Mn-induced inhibition of GLAST expression (#p < 0.05, ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. control; @p < 0.05; Tukey's test following

anova

). Data are expressed as the mean ± SEM (n = 3).

Fig. 6

Fig. 6

Mn inhibits, whereas E2/TX increases expression of GLAST mRNA. (a) Incubation of astrocytes for 6 h with Mn decreased the expression of GLAST mRNA, while treatment of astrocytes for 24 h with E2/TX increased GLAST expression by RT-PCR, semi-quantitative analysis of GLAST mRNA. (b) Treatment of astrocytes for 6 h with Mn decreases GLAST expression, whereas treatment of astrocytes for 24 h with E2 and TX increased GLAST mRNA expression by the real-time PCR analysis. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; Tukey's test following

anova

. Data are expressed as the mean ± SEM (n = 4).

Fig. 7

Fig. 7

Mn and E2/TX exert opposite effects on the expression of astrocytic transforming growth factor β1 (TGF-β1) mRNA. (a) Treatment of astrocytes for 6 h or 24 h with Mn decreased TGF-β1 expression, whereas E2/TX treatment for 24 h increased TGF-β1 expression by RT-PCR analysis. (b) Real time-PCR analysis showed that treatment of astrocytes for 24 h with Mn decreased TGF-β1 mRNA expression, but E2/TX increased TGF-β1 mRNA expression. *p < 0.05, **p < 0.01 ***p < 0.001 vs. control; Tukey's test following

anova

. Data are expressed as the mean ± SEM (n = 4). Experiments were performed in three independent sets of cultures for verification of results.

Fig. 8

Fig. 8

E2/TX attenuates the Mn-induced inhibition of GLAST expression in astrocytes. Pre-treatment of astrocytes for 18 h with E2/TX prior to Mn exposure for 6 h protected against the Mn-induced inhibition of GLAST mRNA expression by RT-PCR (a) and real time-PCR analyses (b) (###p < 0.001 vs. control; *p < 0.05, **p < 0.01 vs. the Mn treatment; Tukey's test following

anova

). Data are expressed as the mean ± SEM (n = 4). Experiments were performed in three independent sets of cultures for verification of results.

Fig. 9

Fig. 9

TGF-β1 increases glutamate uptake in a concentration- and time-dependent manner in astrocytes. (a) Treatment of astrocytes for 24 h with TGF-β1 (2, 4, 8 ng/mL) increased glutamate uptake. (b) TGF-β1 at 4 ng/mL increased glutamate uptake most effectively after 24 h treatment in astrocytes. (c) Pre-treatment with TGF-β1 (18 h) prior to Mn treatment (6 h) reversed the Mn-induced glutamate uptake inhibition in astrocytes. (d) SB431542 (a TGFR inhibitor, 20 μM) abolished the E2/TX-induced enhancement of glutamate uptake (###p < 0.01 vs. control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (a,b,d), or the Mn treatment (c), #p < 0.05; Tukey's test following

anova

). Data are expressed as the mean ± SEM (n = 4).

Fig. 10

Fig. 10

(a,b) Activation of the MAPK/ERK signaling pathway is required for both E2/TX(a)- and TGF-β1(b)-induced enhancement of glutamate uptake in astrocytes. The addition of PD98059 50 μM, an inhibitor of MAPK/ERK, to the culture media 30 min prior to E2/TX or TGF-β1 treatment for 24 h abolished the E2/TX- or TGF-β1-induced enhancement of glutamate uptake (PD98059 was dissolved in DMSO as a concentration of 6.5 mg/mL and diluted with Opti-MEM experimental media). (c,d) Activation of the PI3K/Akt signaling pathway is required for both E2/TX(c)- and TGF-β1(d)-induced enhancement of glutamate uptake in astrocytes. The addition of LY294002 20 μM, an inhibitor of PI3K/Akt, to the culture media 30 min prior to E2/TX or TGF-β1 treatment for 24 h abolished the E2/TX- or TGF-β1-incuded enhancement of glutamate uptake (LY294002 was dissolved in DMSO at a concentration of 25 mg/mL and diluted with Opti-MEM media). The same concentration of DMSO was used as a control. (#p < 0.05, ##p < 0.01, ###p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; Tukey's test following

anova

). Data are expressed as the mean ± SEM (n = 4).

Fig. 11

Fig. 11

Proposed mechanism of E2/TX-induced restoration on Mn-induced astroglial glutamate transporter impairment. E2/TX may act via genomic actions to increase the expression of TGF-β1, which in turn increases GLAST expression in astrocytes. This action is ER-dependent. Modulation of GLAST trafficking by Mn or E2/TX may also play a role in the ability of E2/TX to attenuate Mn-induced glutamate transporter inhibition. Activation of MAPK/ERK and PI3K/Akt signaling is required for E2/TX and TGF-β1 in restoring glutamate transporter function (expression). Glu, glutamate; E2, 17β-estradiol; TX, tamoxifen; ER, estrogen receptor; GLAST, glutamate aspartate transporter; TGFR, transforming growth factor receptor.

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