Selective Activation of Endothelial Cells by the Antioxidant Pyrrolidine Dithiocarbamate: Involvement of C-Jun N-Terminal Kinase and AP-1 Activation

The antioxidant agent pyrrolidine dithiocarbamate (PDTC) has been shown to protect endothelial cells (EC) from pro-inflammatory-induced and pro-oxidant-induced NF-kappaB activation. It also perturbs EC by altering activator protein-1 (AP-1) status and inducing ICAM-1. Experiments were performed to investigate the upstream mechanism by which PDTC produces these effects. We have demonstrated that PDTC not only induced AP-1 binding and ICAM-1 expression by itself, but it also augmented AP-1 activation and ICAM-1 induction in low-dose IL-1alpha treated cells. To dissect the mechanism of these effects, we measured c-Jun and c-Fos expression, and the activity of c-Jun NH2-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) in human umbilical vein endothelial cells (HUVEC). We detected an increase in JNK activity in PDTC-treated HUVEC. Following cotransfection with JNK[K-M], a kinase-deficient JNK1, the PDTC-increased AP-1-driven-luciferase activity was attenuated. Utilizing a specific trans-reporting system we confirmed c-Jun activation by upstream signaling mechanisms. The results show that c-Jun activity was increased 9-fold after PDTC treatment. In addition, PDTC promoted more transient activation in ERK-c-fos. In contrast, PDTC produced sustained JNK-c-Jun activation, which translated into long-lasting ICAM-1 production. These results suggest that an antioxidant may contribute to chronic vascular endothelial activation.