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B Activation and IL-1ß Gene Expression in Cultured Human Epithelial Cells1
Departments of
*
Molecular and Experimental Medicine and
Immunology, The Scripps Research Institute, La Jolla, CA 92037
Recent evidence suggests a novel role of bradykinin (BK) in
stimulating gene transcription. This study examined the effect of BK on
nuclear factor 
B (NF-B) activation and IL-1ß synthesis in human
epithelial cells. Stimulation of A549 cells and primary bronchial
epithelial cells with BK rapidly activated NF-B. BK also increased
the level of secreted immunoreactive IL-1ß in A549 culture
supernatants, an effect that was blocked by actinomycin D and the B2 BK
receptor antagonist HOE-140. The role of NF-B activation in
BK-induced IL-1ß 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 B
enhancers from the IL-1ß gene, while deletion of the B enhancer
sequences eliminated BK-stimulated CAT activity. C3 transferase
exoenzyme, an inhibitor of Rho, abolished BK-induced NF-B activation
at 10 µg/ml and significantly inhibited BK-stimulated IL-1ß
synthesis at 5 µg/ml. A dominant-negative form of RhoA (T19N)
inhibited BK-stimulated reporter gene expression in a dose-dependent
and B-dependent manner. Cotransfection of A549 cells with an
expression vector encoding a constitutively active form of RhoA (Q63L)
along with the IL-1ß promoter-CAT reporter plasmid resulted in a
marked increase in NF-B activity compared with transfection with the
IL-1ß promoter-CAT reporter plasmid alone. These results demonstrate
that BK stimulates NF-B activation and IL-1ß synthesis in A549
cells, and that RhoA is both necessary and sufficient to mediate this
effect.
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