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

Abstract

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 in vivo 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.

Ethanol induces cytostasis of cortical basal progenitors

“…Hence in the current study we used physiologically relevant ETOH concentrations of 2.5 mg/ml and 4 mg/ml corresponding to ~54 mM and ~ 86 mM respectively. ETOH treatments were performed in a separate incubator previously saturated with 100 % (200 proof) ethanol in order to maintain the ETOH concentration at the level added to the media [ 28 ]. Further, ETOH concentration was regularly monitored using Analox AM1 alcohol analyzer (Analox Instruments, MA, USA) [ 29 ].…”

“…Notably, much of the research in FASD has focused on apical progenitors: neural stem cell and radial glia cells and several mechanisms including defective proliferation, disrupted cell cycle events, decreased neurogenesis, differentiation and increased apoptosis have been uncovered explaining the teratogenic effects of ethanol on this population of cells [ 20 – 28 ]. Although the existing few studies on BPs demonstrate an emerging trend about their role in the development of massive cortical surface expansion, gyrification and laminar patterning [ 24 , 25 ], research pertaining to effects of alcohol and the mechanisms underlying altered corticogenesis in BPs are still scant and unclear.…”

Role for Cystathionine γ Lyase (CSE) in an Ethanol (E)-Induced Lesion in Fetal Brain GSH Homeostasis

“…Cells were seeded in six-well plates at a density of 3 × 10 5 cells/well. Following day, cells were transfected with either 20, 50 or 100 nM of siGenome smartpool mix of four Cse specific siRNAs or non-targeting siRNA pool were transfected into rat cortical neuroblasts in serum free condition using endoporter (Genetools, Philomath, OR, USA) [ 4 , 6 , 28 , 68 ]. Prior to transfection, cells were replaced with 800 µL of fresh media and transfected with 200 µL of transfection complex.…”

“…Total protein concentration was determined in the clarified lysates and equal amounts of cellular protein were loaded on 10% or 12% sodium dodecyl sulfate polyacrylamide gel and electrophoresed. The proteins were transferred onto a PVDF membrane and blocked in 5% nonfat dry milk powder (prepared in PBS with 1% Tween) for 1 h. The membranes were then incubated with primary antibodies against CSE, FL-caspase 3, Cl-caspase 3, GAPDH or ACTIN in 5% milk for either 3 h or overnight at 4 °C as previously described [ 28 , 68 ]. The primary antibody-incubated membranes were subsequently washed with PBST for 3 times, incubated with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase in PBST (1:5000 or 1:10,000) for 1 h at room temperature except for CSE blots that were incubated with the secondary antibody in 5% milk, washed with PBST for 5 min × 5 times each.…”

Ethanol (E) Impairs Fetal Brain GSH Homeostasis by Inhibiting Excitatory Amino-Acid Carrier 1 (EAAC1)-Mediated Cysteine Transport

“…The clarified supernatants were estimated for protein concentration and equal amounts of cellular protein were loaded on a 4–12% Bis-Tris gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins were electro-transferred onto a PVDF membrane and blocked with 5% nonfat dry milk powder in PBST for 1 h. The membranes were then incubated with primary antibodies against EAAC1, γ-GT, APN or GAPDH or ACTIN for 3 h or overnight at 4 °C as previously described [ 69 , 80 ] and subsequently washed with PBST for 3 times. Later the membranes were incubated with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (1:5000 or 1:10,000) for 1 h at room temperature, washed with PBST for 5 min × 5 times each, and subjected to ECL-chemiluminescence to detect the bands of proteins.…”