Endoplasmic reticulum (ER) stress inhibitor salubrinal protects against ceramide-induced SH-SY5Y cell death

https://doi.org/10.1016/j.bbrc.2012.08.068Get rights and content

Abstract

In the present study, we examined the mechanisms of ceramide-induced cell death in SH-SY5Y human neuroblastoma cells. Our results demonstrate a significant endoplasmic reticulum (ER) stress response in SH-SY5Y cells after short-chain ceramide (C6) treatment. Administration of ceramide (C6) to SH-SY5Y human neuroblastoma cells caused apoptotic cell death, which was inhibited by ER stress inhibitor salubrinal. Further, ceramide-induced cell death reduced significantly in stable SH-SY5Y cells expressing C/EBP homologous protein (CHOP) shRNA. Salubrinal inhibited ceramide-induced inositol-requiring enzyme 1α (IRE1α)/apoptosis signal regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) phosphorylation. Taken together, these data suggest that ceramide-induced SH-SY5Y cell death may be linked to the ER stress-regulated intrinsic pathway, and proposed the potential protective effects of salubrinal.

Highlights

► Short-chain ceramide (C6) induces ER stress response in SH-SY5Y cells. ► ER stress inhibitor salubrinal protects against ceramide (C6)-induced SH-SY5Y cell death. ► CHOP RNAi knockdown cells are resistant to ceramide (C6). ► Salubrinal inhibits ceramide (C6)-induced IRE1α-ASK1-JNK phosphorylation.

Introduction

Ceramide regulates cell cycle arrest, cell differentiation and apoptosis [1], [2], [3], [4]. Ceramide pathways are involved in a variety of neurological disorders including epilepsy, cerebral ischemia, Alzheimer’s and Parkinson’s diseases [5], [6]. Administration of cell-permeable ceramide analogs or sphingomyelinase causes neuronal cell death in SH-SY5Y human neuroblastoma cells and various neuronal culture cells [7], [8], [9], [10], [11], [12], [13], [14]. Despite growing evidence suggesting a role for ceramide in neuronal apoptosis, specific intracellular pathways of ceramide-induced neuronal death remain to be identified.

Normally, endoplasmic reticulum (ER) regulates the synthesis, post-translational modification and maturation of newly-synthesized proteins. It is also important for maintaining intracellular calcium homeostasis. Various stress conditions disrupt the proper functions of ER and cause ER stress. Receptors at ER membrane including double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1α (IRE1α) act as the sensors of ER stresses. These receptors initiate unfolded protein responses (UPR) [15] to cause: (1) transcriptional up-regulation of ER chaperones (such as C/EBP homologous protein (CHOP); (2) translational attenuation to limit further accumulation of misfolded proteins; and (3) ER-associated degradation to eliminate misfolded proteins [15]. Although the UPR is primarily a pro-survival response, in the event of prolonged or severe ER stress, the UPR switches to cause cell apoptosis [15].

In the present study, we examined the mechanisms of ceramide-induced SH-SY5Y cell death by focusing ER stress pathway. We also tested the potential effects of ER stress inhibitor salubrinal in this condition. Our study provides further evidence in support of a role for ceramide in neuronal apoptosis.

Section snippets

Chemicals and reagents

ER stress inhibitor salubrinal was purchased from Calbiochem (La Jolla, CA). Ceramide (C6) was obtained from Avanti (Alabaster, Alabama). Anti-phospho-IRE1α (Ser 724) was purchased from Novus Biologicals (Littleton, Colorado). Anti-apoptosis signal regulating kinase 1 (ASK1), tubulin, IRE1α antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other phospho-antibodies and their nonphospho-control antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Short-chain ceramide (C6) induces ER stress response in SH-SY5Y cells

We first examined ER stress response in ceramide-treated SH-SY5Y cells by testing phosphorylation of PERK (Thr 980), IRE1α (Ser 724) and the expression of CHOP. Western-blots results in Fig. 1A demonstrated a significant ER stress response after ceramide (short-chain C6) administration in SH-SY5Y cells. Ceramide (C6) induced significant PERK/IRE1α phosphorylation and CHOP expression (Fig. 1A and B). Compared to untreated control level, after 4, 8 and 16 h of ceramide (C6, 25 μM) treatment, PERK

Discussion

The precise role for ceramide in neuronal death and survival remains uncertain. Studies have reported the protective effects of intraventricular injections of C2-ceramide in an immature rat brain hypoxia–ischemia model [18], [19]. However, Yu et al. demonstrated beneficial effects of ceramide downregulation in mice subjected to cerebral ischemia [20]. In cultured rat cerebellar granule cells (CGC) treated with trophic support withdraw or etoposide, ceramide level was increased and was associated

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