The cardioprotective effects of zileuton, a 5-lipoxygenase inhibitor, are mediated by COX-2 via activation of PKCδ

Zileuton has been demonstrated to act as an anti-inflammatory agent by virtue of its well-known ability to inhibit 5-lipoxygenase (5-LO). However, the effects of zileuton on cardiovascular disease and cardiomyocyte apoptosis are unclear. Here, we investigated the effects of zileuton on apoptosis of cardiac myogenic H9c2 cells and neonatal rat cardiomyocytes (NRCMs), and examined the possible role of PKCδ-mediated induction of COX-2 in these effects. Treatment of H9c2 cells with zileuton efficiently induced COX-2 expression and PGE₂ biosynthesis in a time- and dose-dependent manner. Zileuton also exerted a profound protective effect against H₂O₂-induced oxidative stress, a mimic of reperfusion damage in vitro, and this protective effect was abolished by COX-2-selective inhibitor. When we investigated the signalling pathways involved in zileuton-induced COX-2 expression, we found that zileuton acts as a PKCδ activator, causing it to translocate from the cytosol to nucleus. Inhibition of PKCδ activation with rottlerlin, a PKCδ-specific inhibitor, abolished the zileuton-induced protection against H₂O₂-induced cell death and inhibited zileuton-induced COX-2 expression and PGE₂ production. The protective effect of zileuton was dramatically diminished by treatment with LY294002 or PD98059. Furthermore, zileuton-stimulated ERK1/2 and Akt phosphorylation was attenuated by rottlerin, indicating that PKCδ might act upstream of ERK1/2 and Akt. Moreover, inhibition of either ERK1/2 or Akt activation abolished zileuton-induced COX-2 expression. Knockdown of PKCδ with siRNA also reversed the protective effect of zileuton and blocked the induction of COX-2. These results suggest that zileuton-induced COX-2 expression is sequentially mediated through PKCδ-dependent activation of ERK1/2 and Akt. Based on these findings, we propose that zileuton might provide a new therapeutic strategy for ischemia/reperfusion injury of the heart.