A Functional Role of IκB-ε in Endothelial Cell Activation

Abstract

The NF-κB inhibitor IκB-ε is a new member of the IκB protein family, but its functional role in regulating NF-κB-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, IκB-ε associates predominantly with the NF-κB subunit Rel A and to a lesser extent with c-Rel, whereas IκB-α and IκB-β associate with Rel A only. Following stimulation with TNF-α, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented IκB kinase-induced IκB-α, but not IκB-β or IκB-ε phosphorylation and degradation. Since the activation of NF-κB is required for the induction of adhesion molecule expression, we examined the role of IκB-ε in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of κB cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 κB promoter activity. By electrophoretic mobility shift assay, NF-κB heterodimers containing c-Rel specifically bind to the κB motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 κB promoter activity to a greater extent than that of E-selectin and overexpression of IκB-ε inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated IκB-ε is involved in the induction of ICAM-1 expression.