Uncoupling protein-2 up-regulation and enhanced cyanide toxicity are mediated by PPARα activation and oxidative stress

Abstract

Uncoupling protein 2 (UCP-2) is an inner mitochondrial membrane proton carrier that modulates mitochondrial membrane potential (Δ Ψ m) and uncouples oxidative phosphorylation. We have shown that up-regulation of UCP-2 by Wy14,643, a selective peroxisome proliferator-activated receptor-α (PPARα) agonist, enhances cyanide cytotoxicity. The pathway by which Wy14,643 up-regulates UCP-2 was determined in a dopaminergic cell line (N27 cells). Since dopaminergic mesencephalic cells are a primary brain target of cyanide, the N27 immortalized mesencephalic cell was used in this study. Wy14,643 produced a concentration- and time-dependent up-regulation of UCP-2 that was linked to enhanced cyanide-induced cell death. MK886 (PPARα antagonist) or PPARα knock-down by RNA interference (RNAi) inhibited PPARα activity as shown by the peroxisome proliferator response element-luciferase reporter assay, but only partially decreased up-regulation of UCP-2. The role of oxidative stress as an alternative pathway to UCP-2 up-regulation was determined. Wy14,643 induced a rapid surge of ROS generation and loading cells with glutathione ethyl ester (GSH-EE) or pre-treatment with vitamin E attenuated up-regulation of UCP-2. On the other hand, RNAi knockdown of PPARα did not alter ROS generation, suggesting a PPARα-independent component to the response. Co-treatment with PPARα-RNAi and GSH-EE blocked both the up-regulation of UCP-2 by Wy14,643 and the cyanide-induced cell death. It was concluded that a PPARα-mediated pathway and an oxidative stress pathway independent of PPARα mediate the up-regulation of UCP-2 and subsequent increased vulnerability to cyanide-induced cytotoxicity.