Tamoxifen induces protection against manganese toxicity by REST upregulation via the ER-α/Wnt/β-catenin pathway in neuronal cells

Abstract

Chronic exposure to elevated levels of manganese (Mn) causes a neurological disorder referred to as manganism, with symptoms resembling Parkinson's disease (PD). The repressor element-1 silencing transcription factor (REST) has been shown to be neuroprotective in several neurological disorders, including PD, suggesting that identifying REST upregulation mechanisms is an important avenue for the development of novel therapeutics. 17β-estradiol (E2) activates the Wnt/β-catenin signaling, which is known to increase REST transcription. E2 and tamoxifen (TX), a selective estrogen receptor modulator, exerted protection against Mn toxicity. In this study, we tested if TX upregulates REST potentially via Wnt/β-catenin signaling in Cath.a-differentiated (CAD) neuronal cells using luciferase assay, qPCR, Western blot analysis, immunocytochemistry, mutagenesis, chromatin immunoprecipitation, and electrophoretic mobility shift assay. TX (1 μM) increased REST promoter activities and mRNA/protein levels and attenuated Mn (250 μM)-decreased REST transcription in parallel with TX's protective effects against Mn-induced toxicity, potentially via Wnt. TX activated Wnt/β-catenin signaling by preventing β-catenin degradation via inactivation of glycogen synthase kinase-3 beta, leading to increased β-catenin levels and its nuclear translocation and binding to T-cell factor/lymphoid enhancer binding factor sites on Wnt-responsive elements (WRE) of the REST promoter. Mutation of WRE abolished TX-induced REST promoter activity. TX-induced Wnt signaling activation was primarily via the estrogen receptor (ER)-α, although ER-β and G protein-coupled estrogen receptor 1 also mediated TX's action on REST transcription. These findings underscore the critical role of Wnt/β-catenin signaling in TX-induced REST transcription, affording protection mechanisms against Mn toxicity and neurological disorders associated with REST dysfunction.

Keywords: NRSF; REST; Wnt signaling; estrogen receptor; manganese; neuroprotection; tamoxifen; β-catenin.

Figure 1

TX increased REST promoter activity, mRNA, and protein levels in CAD cells.A, CAD neurons were transfected with a human 5′UTR-REST promoter vector and then exposed to 1 μM TX, followed by a luciferase assay to measure REST promoter activity. B and C, CAD cells were treated with 1 μM TX, then followed by measurement of REST mRNA using qPCR (B) and REST protein using Western blot (C). GAPDH and β-actin were used as loading controls of mRNA and protein, respectively. ∗∗∗p < 0.001, ∗∗p < 0.01 compared to control. (One-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 2

TX increased REST expression mainly via ER-α in CAD cells.A, CAD cells were transfected with GFP-tagged empty vector (EV) control, ER-α, ER-β, and GPER1 vectors. Fluorescent images of CAD cells were captured after transfection, and fluorescence intensity was quantified (scale bar, 200 μM). B, CAD neurons were co-transfected with human REST promoter and either EV, ER-α, ER-β, or GPER1 expression vectors, then treated with 1 μM TX for 6 h, followed by luciferase assay to detect REST promoter activity. C and D, Transfected CAD cells were treated with 1 μM TX for 12 h and 24 h to determine REST mRNA (C) and REST protein (D), respectively. GAPDH and β-actin were used as loading controls of mRNA and protein, respectively. ^@@@p < 0.001, ^@@p < 0.01, ^@p < 0.05 compared to control of each group. (One-way ANOVA followed by Tukey's post hoc; n = 3). The data shown are representative of 3 independent experiments.

Figure 3

TX activated the ER-α/Wnt/β-catenin signaling pathway in CAD cells.A, CAD were transfected with EV or ER-α vectors, then treated with 1 μM TX, followed by measurement of ER-α and β-catenin protein using Western blot. CAD neurons were treated with 1 μM TX for several time points, followed by ER-α and β-catenin mRNA levels detection using qPCR. B, CAD cells were treated with ER-α antagonist MPP for 6 h, followed by measurement of β-catenin protein. C, CAD cells were treated with MPP for 6 h prior to 6 h exposure with TX (in the presence of MPP), then subsequently measured β-catenin protein levels. β-actin was used as a loading control of protein. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, ^###p < 0.001, ^#p < 0.05 compared to control. ^@@@p < 0.001 compared to each other. (One-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 4

TX attenuated Mn-induced cytotoxicity as well as Mn-decreased REST expression in CAD cells.A, CAD cells were treated with 1 μM TX for 6 h prior to 250 μM Mn exposure for 2 h (in the presence of TX), followed by measurement of ROS levels by a fluorometer using CM-H₂DCFDA. B, CAD cells were treated with 1 μM TX for 24 h, followed by 250 μM Mn exposure for 24 h (in the presence of TX), followed by assessment of cell viability using MTT assay. C, CAD cells were treated with 3 μM LGK-974 for 12 h, followed by 1 μM TX for 12 h, then exposed to 250 μM Mn for 24 h (in the presence of LGK-974 and TX). Subsequently, cell viability was assessed using an MTT assay. D, CAD cells were transfected with a human 5′UTR-REST promoter vector, then treated with 1 μM TX for 6 h prior to 250 μM Mn exposure for 6 h (in the presence of TX), followed by measurement of REST promoter activity. E, CAD were treated with 1 μM TX for 12 h prior to 250 μM Mn exposure for 12 h (in the presence of TX), followed by measurement of REST mRNA. F, CAD were treated with TX for 24 h, followed by 250 μM Mn for 24 h (in the presence of TX), followed by measurement of REST protein. GAPDH and β-actin were used as loading controls of mRNA and protein, respectively. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, ^###p < 0.001, ^##p < 0.01, ^#p < 0.05 compared to control. ^@@@p < 0.001, ^@@p < 0.01 compared to each other. (One-way ANOVA followed by Tukey's post hoc; n = 3). The data shown are representative of 3 independent experiments.

Figure 5

TX increased Wnt3a and attenuated Mn-induced Wnt3a downregulation in CAD cells.A and B, CAD cells were treated with 1 μM TX (A), and 250 μM Mn (B), followed by measurement of Wnt3a protein and mRNA using Western blot and qPCR, respectively. C, CAD were treated with 1 μM TX for 1 h prior to 250 μM Mn for 3 h (in the presence of TX), followed by measurement of Wnt3a protein. β-actin and GAPDH were used as loading controls for protein and mRNA, respectively. ∗∗∗p < 0.001, ^###p < 0.001, ^##p < 0.01, ^#p < 0.05 compared to control. ^@@@p < 0.001 compared to the Mn-treated group. (One-way ANOVA followed by Tukey's post hoc; n = 3). The data shown are representative of 3 independent experiments.

Figure 6

TX decreased Dkk-1 and attenuated Mn-induced Dkk-1 upregulation in CAD.A and B, CAD cells were treated with 1 μM TX (A), and 250 μM Mn (B), followed by measurement of Dkk-1 protein and mRNA using Western blot and qPCR, respectively. C, CAD cells were treated with 1 μM TX for 1 h prior to 250 μM Mn for 3 h (in the presence of TX), followed by measurement of Dkk-1 protein. β-actin and GAPDH were used as loading controls for protein and mRNA, respectively. ∗∗∗p < 0.001, ∗p < 0.05, ^###p < 0.001, ^##p < 0.01 compared to control. ^@@@p < 0.001 compared to the Mn-treated group. (One-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 7

TX blocked Mn-induced activation of GSK3β by modulating phosporylation at the Y216 site.A and B, CAD cells were treated with 1 μM TX (A) and 250 μM Mn (B), followed by measurement of phosphorylation of Y216 on GSK3β (p-GSK3β Y216) using Western blot. C, CAD were treated with 1 μM TX for 1 h prior to 250 μM Mn exposure for 1 h (in the presence of TX), followed by measurement of p-GSK3β Y216 protein levels using Western blot. β-actin was used as a loading control of protein. ∗∗p < 0.01, ∗p < 0.05, ^###p < 0.001, ^##p < 0.01 compared to control. ^@@@p < 0.001 compared to Mn-treated group. (One-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 8

TX increased, while Mn decreased, inactive GSK3β by modulating phosphorylation at the S9 site.A and B, CAD cells were treated with 1 μM TX (A), and 250 μM Mn (B), followed by measurement of phosphorylation of S9 on GSK3β (p-GSK3β S9). β-actin was used as a loading control of protein. ∗∗p < 0.01, ∗p < 0.05, ^###p < 0.001 compared to control. (One-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 9

TX increased nuclear translocation of β-catenin and its interaction with TCF/LEF.A, CAD cells were exposed to 250 μM Mn, followed by measurement of phosphorylation of β-catenin on S33, S37, and T41 residues, along with the total β-catenin protein levels using Western blot. B, CAD cells were treated with 1 μM TX for 1 h prior to 250 μM Mn exposure for 6 h (in the presence of TX), followed by measurement of total β-catenin protein. C, immunofluorescence images of β-catenin nuclear translocation upon incubation of 1 μM TX for 3 h prior to 250 μM Mn treatment for 6 h (in the presence of TX) in CAD neurons (scale bar, 10 μM). D, Nuclear β-catenin protein levels were measured from nuclear extracts from CAD cells after treatment of 1 μM TX for 3 h, followed by Mn exposure for 6 h (in the presence of TX) using Western blot. E, Nuclear extracts from TX-treated CAD neurons were pulled down using β-catenin antibody, followed by quantification and assessment of its interaction with WRE-bound TCF/LEF TF. β-actin was used as a loading control of total protein extract and histone H3 for nuclear fraction. ∗∗∗p < 0.001, ^###p < 0.001, ^##p < 0.01, compared to control. ^@@@p < 0.001 compared to Mn-treated group. (Student's t test or one-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 10

TX attenuated Mn-induced decrease of β-catenin binding to the WRE of the REST promoter.A, illustration of the human 5′ UTR (−3390/+1) REST promoter region with WREs. B, after CAD cells were treated with 1 μM TX for 12 h, followed by 250 μM Mn exposure for 24 h (in the presence of TX), ChIP assay was performed to determine the binding of β-catenin on the WREs on the REST promoter in vivo, followed by quantification of β-catenin-bound DNA by real-time qPCR. C, EMSA was performed in nuclear extracts prepared from CAD cells treated with 1 μM TX for 12 h, followed by 250 μM Mn exposure for 24 h (in the presence of TX) as described in the Experimental procedures. The black arrow shows the DNA–protein complex, which was quantified using ImageLab Software (BioRad). D, two site mutations of the WRE consensus sequences are indicated in red. E, CAD neurons were transfected overnight with wild-type human 5′ UTR REST promoter (REST WT) or WRE mutants of REST promoter plasmid (REST WRE mut), then subsequently treated with 1 μM TX for 6 h, followed by luciferase assay. ∗∗∗p < 0.001, ∗∗p < 0.01, ^##p < 0.01, compared to control. ^@@@p < 0.001, ^@@p < 0.001 compared to each other. (One-way ANOVA followed by Tukey's post hoc, n = 3). The data shown are representative of 3 independent experiments.

Figure 11

Schematic diagram of the proposed mechanism of TX's neuroprotection against Mn toxicity by upregulating REST expression via Wnt/β-catenin signaling in CAD neurons. TX activates the Wnt/β-catenin signaling pathway via ER-α, while Mn inhibits this pathway by dysregulating Wnt3a and Dkk-1. TX and Mn modulate GSK3β function by differentially phosphorylating GSK3β at S9 and/or Y216, leading to alteration of β-catenin levels in the cytosol and subsequent translocation to the nucleus. β-catenin forms a complex with TCF/LEF in the nucleus, which binds to WRE on the REST promoter, inducing REST transcription. These findings suggest that TX exerts neuroprotective effects against Mn toxicity by increasing REST via the ER-α/Wnt/β-catenin signaling pathway. Red: Mn-induced effects; blue: TX-induced effects.