Ethanol-Induced Transcriptional Activation of Programmed Cell Death 4 (<i>Pdcd4</i>) Is Mediated by GSK-3β Signaling in Rat Cortical Neuroblasts

<div><p>Ingestion of ethanol (ETOH) during pregnancy induces grave abnormalities in developing fetal brain. We have previously reported that ETOH induces programmed cell death 4 (PDCD4), a critical regulator of cell growth, in cultured fetal cerebral cortical neurons (PCNs) and in the cerebral cortex <i>in vivo</i> and affect protein synthesis as observed in Fetal Alcohol Spectrum Disorder (FASD). However, the mechanism which activates PDCD4 in neuronal systems is unclear and understanding this regulation may provide a counteractive strategy to correct the protein synthesis associated developmental changes seen in FASD. The present study investigates the molecular mechanism by which ethanol regulates PDCD4 in cortical neuroblasts, the immediate precursor of neurons. ETOH treatment significantly increased PDCD4 protein and transcript expression in spontaneously immortalized rat brain neuroblasts. Since PDCD4 is regulated at both the post-translational and post-transcriptional level, we assessed ETOH’s effect on PDCD4 protein and mRNA stability. Chase experiments demonstrated that ETOH does not significantly impact either PDCD4 protein or mRNA stabilization. PDCD4 promoter-reporter assays confirmed that PDCD4 is transcriptionally regulated by ETOH in neuroblasts. Given a critical role of glycogen synthase kinase 3β (GSK-3β) signaling in regulating protein synthesis and neurotoxic mechanisms, we investigated the involvement of GSK-3β and showed that multifunctional GSK-3β was significantly activated in response to ETOH in neuroblasts. In addition, we found that ETOH-induced activation of PDCD4 was inhibited by pharmacologic blockade of GSK-3β using inhibitors, lithium chloride (LiCl) and SB-216763 or siRNA mediated silencing of GSK-3β. These results suggest that ethanol transcriptionally upregulates PDCD4 by enhancing GSK-3β signaling in cortical neuroblasts. Further, we demonstrate that canonical Wnt-3a/GSK-3β signaling is involved in regulating PDCD4 protein expression. Altogether, we provide evidence that GSK-3β/PDCD4 network may represent a critical modulatory point to manage the protein synthetic anomalies and growth aberrations of neural cells seen in FASD.</p></div

Chemical inhibition of GSK-3β signaling pathway increases PDCD4 expression.

<p>Cells were pre-treated with with LiCl (10 mM) or SB-216763 (20 µM) for 1 h and then exposed with ETOH for 12 h. Panel A shows Western blot and densitometric scanning analysis of PDCD4 and tubulin with LiCl and ETOH treatment. (<b>B</b>) At the end of LiCl and ETOH treatment, cells were processed for qRT-PCR analysis to determine PDCD4 mRNA expression. Results are expressed as fold change in mRNA relative to GAPDH control. (<b>C</b>) Cells were transfected with either promoterless, pGL4.16 plasmid or 1046 bp PD PROM construct for 24 h. Following transfection, cells were pretreated with LiCl for 1 h and then exposed with ETOH for 12 h. After treatment, cells were processed for determining luciferase activity. Data are expressed as fold change relative to control. <b>D</b>, <b>E</b> and <b>F</b> depicts the Western blot analysis, RT-PCR analysis (expressed in fold change) and relative luciferase activity (expressed in fold change) when treated with SB-216763, a known GSK-3β inhibitor. In A–F, statistical analysis was performed using one-way ANOVA followed by Newman-Keul’s posthoc correction. *is statistically significant at p<0.05. n = 6.</p

Ethanol activates GSK-3β.

<p>Neuroblasts were treated with ETOH for 2, 4, 8, 12 and 24 h. Protein levels of phospho-GSK-3β (Ser 9) were determined in control and ETOH treated cells by Western blot analysis (top). Statistical significance was evaluated by normalizing with GSK-3β and GAPDH (bottom). Analysis was performed using one-way ANOVA followed by Newman-Keul’s posthoc test. *denotes p<0.05 compared with control. n = 3.</p

Ethanol time and dose dependently increases PDCD4 expression in cortical neuroblasts.

<p>(<b>A</b>) Cells were treated with or without ETOH (4 mg/ml) for 4, 8, 12 and 24 h. PDCD4 levels in neuroblasts were evaluated by immunoblot analysis. Anti-tubulin was used to demonstrate equal loading. Lower panel shows densitometric scanning analysis ratio of PDCD4 to tubulin. (<b>B</b>) Cells were treated with 1 mg/ml (∼21 mM), 2.5 mg/ml (∼54 mM) and 4 mg/ml (∼86 mM) ethanol for 24 h. Cells were then harvested and immunoblotted with anti-PDCD4 and anti-GAPDH. Protein levels were quantitated using Scion Image software. One-way analysis of variance (ANOVA) and Newman-Keul’s posthoc test was performed to establish statistical significance.*p<0.05 when compared with untreated control (A & B), n = 3.</p

Ethanol exposure induces PDCD4 independent of mRNA stability in neuroblasts.

<p>(<b>A</b>) Quantitative real time PCR was performed on mRNA obtained from neuroblasts treated with or without ETOH (4 mg/ml) for indicated time periods. <i>Pdcd4</i> expression was determined after normalizing to GAPDH mRNA expression. Statistical significance was established by one-way ANOVA and Newman-Keul’s posthoc test.*p<0.05 when compared to control. (<b>B & C</b>) Cells were treated with or without 4 mg/ml of ETOH followed by Act D (1 µg/ul) treatment for indicated time periods (4, 8, 12 and 24 h) and cells were harvested at the end of the experiments for either PDCD4 or GAPDH mRNA analysis by qRT-PCR (<b>B</b>) or protein analysis by Western blotting (<b>C</b>). Statistical analysis was carried out by two-way analysis of variance (ANOVA) with Bonferroni post hoc tests.*p<0.05 compared with control. n = 3.</p

Ethanol transcriptionally upregulates <i>Pdcd4</i>.

<p>(<b>A</b>) Rat <i>Pdcd4</i> gene structure derived from GenBank (Accession No. BC167751). <i>Pdcd4</i> consists of 12 exons (E1–E12) out of which E2–E12 are coding exons and E1 is a non-coding exon which forms the 5′ untranslated region. Sequences 1046 bp upstream of the transcriptional start site (TSS) regarded as <i>Pdcd4</i> promoter was used in the present study. The transcriptional start site is designated as +1 and CpG island is represented by the horizontal bar above E1 of rat <i>Pdcd4</i> gene. (<b>B</b>) Cells were transfected with either pGL4.16 or PD PROM constructs and luciferase activity was evaluated 24 h post-transfection using dual luciferase reporter assay system. Data was analyzed using Student’s t-test. *p<0.001. (<b>C</b>) Neuroblasts were transfected with either pGL4.16, promoterless or PD PROM (−1046) for 24 h. Following transfection, cells were treated with or without 4 mg/ml of ETOH for 12 h and were processed for luciferase activity. Data was analyzed using one-way ANOVA and Newman-Keul’s posthoc test.*p<0.001. n = 6.</p