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

Uncoupling protein 2 (UCP-2) is an inner mitochondrial membrane proton carrier that modulates mitochondrial membrane potential (A psi(m)) and uncouples oxidative phosphorylation. We have shown that up-regulation of UCP-2 by Wyl4,643, a selective peroxisome proliferator-activated receptor-alpha (PPAR alpha) agonist, enhances cyanide cytoloxicity. The pathway by which Wyl4,643 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 (PPAR alpha antagonist) or PPARa knock-down by RNA interference (RNAi) inhibited PPAR(X 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 PPARa did not alter ROS generation, suggesting a PPAR alpha-independent component to the response. Co-treatment with PPAR alpha-RNAi and GSH-EE blocked both the up-regulation of UCP-2 by Wy 14,643 and the cyanide-induced cell death. It was concluded that a PPAR alpha mediated pathway and an oxidative stress pathway independent of PPAR alpha mediate the up-regulation of UCP-2 and subsequent increased vulnerability to cyanide-induced cytotoxicity. (c) 2007 Elsevier Inc. All rights reserved.