Involvement of PGE2 and PGDH but not COX-2 in thrombin-induced cortical neuron apoptosis

Abstract

The pathways that contribute to thrombin-induced neuron death have been incompletely defined. Induction of cyclooxygenase 2 (COX-2), the enzyme that catalyzes the first step in prostaglandin synthesis, promotes neuronal injury. PGE2, a downstream product of COX-2 metabolism, is neurotoxic in vitro and in vivo, and is thought to be the bioactive mediator responsible for COX-2 neurotoxicity. The objective of this study is to determine the ability of thrombin to affect PGE2 metabolism in cultured neurons. The data show that in thrombin-induced apoptosis of cultured neurons, PGE2 release increases when COX-2 is absent, and is regulated by prostaglandin dehydrogenase (PGDH), a key enzyme that degrades PGE2. NS398, a COX-2 specific inhibitor, protects neurons against thrombin toxicity, by inducing active PGDH. These data implicate PGDH in thrombin-mediated neuronal cell death.

Figure 1

Western blot analysis of COX-2 expression in cortical neurons induced by thrombin. Cultured neurons were exposed to a range of thrombin concentrations (0–300 nM) for 24 h. Cells were rinsed and lysed and lysates (25 μg) run on SDS-PAGE gel and western blot analysis performed using antibodies to thrombin. Antibodies to GAPDH were used to confirm loading equivalency between lanes. The blot is representative of 3 separate experiments.

Figure 2

Effect of thrombin and NS398 on PGE2 levels in cultured neurons. Neurons in treatment media were treated with thrombin (100 nM), NS398 (100 μM) or both and incubated for 3 h, the media were then collected and analyzed for PGE2 by ELISA. The results are means ± SEM for 3 experiments performed in triplicate. ** p<0.01vs.control ### p<0.05 vs. thrombin alone

Figure 3

Western blot analysis of PGDH in neurons regulated by NS398 and thrombin. Cultured neurons in treatment media were treated with thrombin (100 nM), NS398 (100 μM) or both for 2 h, protein lysed and analyzed by western blotting for Prostaglandin Dehydrogenase (PGDH). The housekeeping gene GAPDH was used as a loading control. Results are means ± SEM for 3 experiments. ***p<0.001 vs control; n=3.