Generation of Cellular Reactive Oxygen Species by Activation of the EP2 Receptor Contributes to Prostaglandin E2-Induced Cytotoxicity in Motor Neuron-Like NSC-34 Cells

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease characterized by progressive degeneration of motor neurons in the central nervous system. Prostaglandin E2 (PGE2) plays a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. We have shown previously that PGE2 directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, a motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) and examined the effects of N-acetylcysteine (NAC), a cell-permeable antioxidant, on PGE2-induced cell death in differentiated NSC-34 cells. Dichlorofluorescein (DCF) fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by a selective EP2 antagonist (PF-04418948) but not a selective EP3 antagonist (L-798,106). Although an EP2-selective agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the level of intracellular ROS in these cells. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with NAC in a concentration-dependent manner. Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Moreover, dibutyryl-cAMP treatment of differentiated NSC-34 cells caused intracellular ROS generation and cell death. Our data reveal the existence of a PGE2-EP2 signaling-dependent intracellular ROS generation pathway, with subsequent activation of the caspase-3 cascade, in differentiated NSC-34 cells, suggesting that PGE2 is likely a key molecule linking inflammation to oxidative stress in motor neuron-like NSC-34 cells.

Highly Efficient Conversion of Motor Neuron-Like NSC-34 Cells into Functional Motor Neurons by Prostaglandin E2

“…Nevertheless, the treatment with PGE 2 for 7 days resulted in a drastic decrease in cell viability. Our laboratory has previously reported that PGE 2 induced cell death in differentiated NSC-34 cells [ 13 , 14 , 23 ]. Therefore, the PGE 2 -induced cell death observed in this study was considered to be consistent with these results.…”

“…The cell viability of NSC-34 cells was determined by an LDH assay using an LDH-Cytotoxic Test Wako, as previously described [ 20 , 23 ]. LDH release was assessed according to the manufacturers’ instructions.…”

The Molecular Mechanisms Underlying Prostaglandin D2-Induced Neuritogenesis in Motor Neuron-Like NSC-34 Cells

“…The Western blot analysis was performed as described previously [26,27]. Briefly, NSC-34 cells were lysed in a radioimmunoprecipitation buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 5 mM EDTA, phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA), and protease inhibitor cocktail (Roche, Basel, Switzerland).…”

“…The amount of viable and dead cells was measured by LIVE/DEAD ® Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Eugene, OR, USA) according to a previously described method [27]. The images were collected with an inversed fluorescence microscope (IX70, Olympus, Tokyo, Japan) and cell mortality was determined by calculating the percentage of EthD-1-positive cells of the total number of cells (the sum of calcein-positive live cells and EthD-1-positive dead cells).…”

The Novel Regulatory Role of lncRNA-miRNA-mRNA Axis in Amyotrophic Lateral Sclerosis: An Integrated Bioinformatics Analysis

“…Amyotrophic lateral sclerosis (ALS) is an incurable chronic neurological disease that can lead to the continuous death of upper and lower motor neurons, resulting in muscle atrophy and fatigue that affect the patient's limb movement until the death of the patient [1][2][3][4]. This disease is most likely to occur in middle-aged people, with an incidence rate of 1.5-2.7 per 100,000 people observed; in addition, most patients die within 5 years after the onset of the disease, which has a severe impact on the physical and mental health of patients [1,5,6].…”