GSK-3β-mediated regulation of cadmium-induced cell death and survival

Abstract

Background: Previous studies indicated that cadmium (Cd) increases PI3-kinase/Akt phosphorylation, resulting in an alteration in GSK-3β activity. However, the mechanism of Cd-induced endoplasmic reticulum (ER) stress in neuronal cells has yet to be studied in needs further elucidation. We examined the role of GSK-3β in Cd-induced neuronal cell death and the related downstream signaling pathways.

Methods: SH-SY5Y human neuroblastoma cells were treated with 10 or 20 μM BAPTA-AM and 1 μM wortmannin for 30 min and then incubated with 25 μM Cd for 12 h. Apoptotic cells were visualized via DAPI and PI staining. Data were evaluated with one-way analysis of variance (ANOVA) followed by Student's t-test. Data are expressed as the means ± SD of experiments performed at least three times.

Results: Treatment of human neuronal SH-SY5Y cells with Cd induced ER, stress as evidenced by the increased expression of GRP78, which is a marker of ER stress. Cd exposure significantly increased the phosphorylation of Akt at thr308 and ser473 and that of GSK-3β at ser9 in a time-dependent manner, while the total protein levels of GSK-3β and Akt did not change. Cd-induced apoptosis was higher in GSK-3β-knockdown cells than in normal cells.

Conclusions: Our data suggest that Akt/GSK-3β signaling activated by Cd is involved in neuronal cell survival.

Keywords: Cadmium; ER-stress; GSK-3β.

Fig. 1

Cadmium induces ER stress in human neuronal cells. SH-SY5Y cells were treated with 25 μM Cd or1 μM thapsigargin for the indicated times. Total cellular proteins were extracted and separated using SDS-PAGE. Western blot analysis was performed using anti-GRP78, anti-GRP94 and anti-GADD153 antibodies

Fig. 2

Cadmium increases intracellular Ca²⁺ concentration in neuronal cells. SH-SY5Y cells were treated with or without 10 μM BAPTA-AM for 30 min and then incubated with 25 μM Cd for the indicated times. a – Ca²⁺ concentration was quantified using the calcium indicator dye, Fluo-4 AM. b – GRP78 was analyzed via western blotting after 30 min pretreatment with 10 μM BAPTA-AM, followed by 12 h incubation with 25 μM Cd. c – Apoptotic cells were counted using the annexin V assay. The data are expressed as the means ± SEM of the apoptotic cells from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Akt and GSK-3β regulation by cadmium. SH-SY5Y cells were treated with 25 μM Cd for the indicated times. Total cellular proteins were extracted and separated using SDS-PAGE. a – Western blot analysis was performed using anti-phospho-Akt (T308), S473 and phospho-GSK-3β (S9) antibodies. Band intensities were quantified based on densitometric values using Fujifilm Science Lab 97 Image Gauge software (version 2.54). The data are from at least three independent experiments. b, c – p-AKT and p-GSK-3β were analyzed via western blotting after 30 min pretreatment with 10 μM BAPTA-AM (b) or 1 μM wortmannin (c), followed by 12 h incubation with 25 μM Cd. d – Apoptotic cells were counted using the annexin V assay. The data are expressed as the means ± SEM of the percentage of apoptotic cells from at least three independent experiments. *p < 0.05, **p < 0.01

Fig. 4

Cadmium-induced apoptosis was increased by GSK-3β knockdown. SH-SY5Y cells were transfected with GSK-3β siRNA, and total proteins were extracted and separated using SDS-PAGE. a – Representative FACS data for apoptosis are shown. b – Western blot analysis was performed using anti-GSK-3β antibodies (inset). Apoptotic cells were counted using the annexin V assay. The data are expressed as the means ± SEM of the percentage of apoptotic cells from at least three independent experiments. ***p < 0.001

Fig. 5

Scheme of the proposed pathways mediating calcium-induced apoptosis. Cd induces ER stress that triggers the pro-apoptotic pathway, including the CHOP and JNK pathways. By contrast, Cd also causes elevation of intracellular Ca²⁺ that contributes to the anti-apoptotic signal via phosphorylation of Akt/GSK3β, and counteracts pro-apoptotic signals