ROLE OF THE RHO-GTPASE IN BRADYKININ-STIMULATED NUCLEAR FACTOR-KAPPA-B ACTIVATION AND IL-1-BETA GENE-EXPRESSION IN CULTURED HUMAN EPITHELIAL-CELLS

Recent evidence suggests a novel role of bradykinin (BK) in stimulatin g gene transcription, This study examined the effect of BK on nuclear factor kappa B (NF-kappa B) activation and IL-1 beta synthesis in huma n epithelial cells, Stimulation of A549 cells and primary bronchial ep ithelial cells with BK rapidly activated NF-kappa B. BK also increased the level of secreted immunoreactive IL-1 beta in A549 culture supern atants, an effect that was blocked by actinomycin D and the B2 BK rece ptor antagonist HOE-140, The role of NF-kappa B activation in BK-induc ed IL-1 beta synthesis was demonstrated by the ability of BK to stimul ate increased chloramphenicol acetyltransferase (CAT) activity in A549 cells transfected with a reporter plasmid containing three kappa B en hancers from the IL-1 beta gene, while deletion of the kappa B enhance r sequences eliminated BK-stimulated CAT activity, C3 transferase exoe nzyme, an inhibitor of Rho, abolished BK-induced NF-kappa B activation at 10 mu g/ml and significantly inhibited BK-stimulated IL-1 beta syn thesis at 5 mu g/ml. A dominant-negative form of RhoA (T19N) inhibited BK-stimulated reporter gene expression in a dose-dependent and kappa B-dependent manner, Cotransfection of A549 cells with an expression ve ctor encoding a constitutively active form of RhoA (Q63L) along with t he IL-1 beta promoter-CAT reporter plasmid resulted in a marked increa se in NF-kappa B activity compared with transfection with the IL-1 bet a promoter-CAT reporter plasmid alone, These results demonstrate that BK stimulates NF-kappa B activation and IL-1 beta synthesis in A549 ce lls, and that RhoA is both necessary and sufficient to mediate this ef fect.