Role of the Rho GTPase in Bradykinin-Stimulated Nuclear Factor-κB Activation and IL-1β Gene Expression in Cultured Human Epithelial Cells

摘要

Recent evidence suggests a novel role of bradykinin (BK) in stimulating gene transcription. This study examined the effect of BK on nuclear factor κB (NF-κB) activation and IL-1β synthesis in human epithelial cells. Stimulation of A549 cells and primary bronchial epithelial cells with BK rapidly activated NF-κB. BK also increased the level of secreted immunoreactive IL-1β in A549 culture supernatants, an effect that was blocked by actinomycin D and the B2 BK receptor antagonist HOE-140. The role of NF-κB activation in BK-induced IL-1β synthesis was demonstrated by the ability of BK to stimulate increased chloramphenicol acetyltransferase (CAT) activity in A549 cells transfected with a reporter plasmid containing three κB enhancers from the IL-1β gene, while deletion of the κB enhancer sequences eliminated BK-stimulated CAT activity. C3 transferase exoenzyme, an inhibitor of Rho, abolished BK-induced NF-κB activation at 10 μg/ml and significantly inhibited BK-stimulated IL-1β synthesis at 5 μg/ml. A dominant-negative form of RhoA (T19N) inhibited BK-stimulated reporter gene expression in a dose-dependent and κB-dependent manner. Cotransfection of A549 cells with an expression vector encoding a constitutively active form of RhoA (Q63L) along with the IL-1β promoter-CAT reporter plasmid resulted in a marked increase in NF-κB activity compared with transfection with the IL-1β promoter-CAT reporter plasmid alone. These results demonstrate that BK stimulates NF-κB activation and IL-1β synthesis in A549 cells, and that RhoA is both necessary and sufficient to mediate this effect.