Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis

https://doi.org/10.1016/j.bbrc.2004.11.173Get rights and content

Abstract

Cd induces oxidative stress and apoptosis in various cells by activating mitogen-activated protein kinases (MAPKs), but the precise signaling components of the MAPK cascade and their role in neuronal apoptosis are still unclear. Here, we report that Cd treatment of SH-SY5Y cells caused apoptosis through sequential phosphorylation of the apoptosis signal regulating kinase 1, MAPK kinase 4, c-Jun N-terminal kinase (JNK), and c-Jun as determined by overexpression of dominant negative (DN) constructs of these genes or using a specific JNK inhibitor SP600125. Both Cd-induced JNK and c-Jun phosphorylation and apoptosis were inhibited dramatically by N-acetyl-l-cysteine, a free radical scavenger. In addition, caspase inhibitors, zDEVD and zVAD, reduced apoptosis but not JNK and c-Jun phosphorylation induced by Cd, while overexpression of DN JNK1 inhibited caspase-3 activity. Taken together, our data suggested that the JNK/c-Jun signaling cascade plays a crucial role in Cd-induced neuronal cell apoptosis and provides a molecular linkage between oxidative stress and neuronal apoptosis.

Section snippets

Materials and methods

Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics were obtained from Gibco-BRL (Gland Island, NY). The MAPK inhibitors, SB203580, U0126, and SP600125 and the caspase inhibitors, z-Val-Ala-Asp-CH2F (zVAD-fmk) and z-ASP (OCH3)-Glu (OCH3)-Val-Asp-CH2F (zDEVD-fmk), were obtained from Calbiochem–Novabiochem (San Diego, CA). Anti-JNK, p38, ERK, c-Jun, phospho-JNK (Thr-183/Tyr-185), phospho-ERK (Thr-202/Tyr-204), phospho-c-Jun (Ser-63), and caspase-3

Cd induced apoptosis of neuronal cells through phosphorylation of JNK

To assess the toxic effects of Cd on human neuronal cell, SH-SY5Y cells were treated with Cd for 12 h at various concentrations (0–100 μM) and assayed for cell viability using the MTT assay, DAPI staining, and TUNEL staining. Loss of viability occurred in a dose-dependent manner in response to Cd treatment (Fig. 1A). At 25 μM Cd, the cell viability reduced to about 54.1 ± 3.4% of the untreated control cells. Phase-contrast microscopic observation showed the damaged cells which were more rounded in

Discussion

Cd-induced apoptosis was described previously in various cells including neuronal cell [5], [15], [23], [29] but its underlying molecular mechanism was not clearly elucidated. In the present study, we used SH-SY5Y cell, a human neuroblastoma cell line, in order to examine the intracellular signaling pathways involved in Cd-induced neuronal cell apoptosis. We found that Cd induced apoptosis as determined by TUNEL assay and DAPI staining, and that the apoptosis occurs through activation of the

Acknowledgments

The authors thank Dr. S.Y. Suh for her valuable comments and Mrs. S.Y. Hur for her assistance in preparing the manuscript. This study was supported by “The Eco-technopia 21 project” from Ministry of Environment for Drs. S.A. Jo and C.K. Moon.

References (40)

  • J. Ruffels et al.

    Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death

    Eur. J. Pharmacol.

    (2004)
  • I. Jo et al.

    Serum deprivation increases the expression of low density lipoprotein receptor-related protein in primary cultured rat astrocytes

    Biochem. Biophys. Res. Commun.

    (2002)
  • K. Domanska-Janik et al.

    Neuroprotection by cyclosporin A following transient brain ischemia correlates with the inhibition of the early efflux of cytochrome C to cytoplasm

    Brain Res. Mol. Brain Res.

    (2004)
  • A. Galan et al.

    Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells

    J. Biol. Chem.

    (2000)
  • J.L. Kummer et al.

    Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase

    J. Biol. Chem.

    (1997)
  • K. Mackay et al.

    An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia

    J. Biol. Chem.

    (1999)
  • J. Cao et al.

    Distinct requirements for p38alpha and c-Jun N-terminal kinase stress-activated protein kinases in different forms of apoptotic neuronal death

    J. Biol. Chem.

    (2004)
  • A.H. Poliandri et al.

    Cadmium induces apoptosis in anterior pituitary cells that can be reversed by treatment with antioxidants

    Toxicol. Appl. Pharmacol.

    (2003)
  • A. Lemarie et al.

    Cadmium induces caspase-independent apoptosis in liver Hep3B cells: role for calcium in signaling oxidative stress-related impairment of mitochondria and relocation of endonuclease G and apoptosis-inducing factor

    Free Radic. Biol. Med.

    (2004)
  • S.J. Stohs et al.

    Oxidative mechanisms in the toxicity of chromium and cadmium ions

    J. Environ. Pathol. Toxicol. Oncol.

    (2001)
  • Cited by (0)

    Abbreviations: Cd, cadmium; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p38, p38 MAP kinase; MKK4/7, MAP kinase kinase 4/7; ASK-1, apoptosis signal regulating kinase; DN, dominant negative.

    View full text