Requirement of phosphatidylinositol 3-kinase activity for bradykinin stimulation of NF-kappa B activation in cultured human epithelial cells

The signaling mechanisms utilized by bradykinin (BK) to activate the transc ription factor nuclear factor kappa B (NF-kappa B) are poorly defined. We p reviously demonstrated that BK-stimulated NF-kappa B activation requires th e small GTPase RhoA, We present evidence that BK-induced NF-kappa B activat ion both activates and requires phosphatidylinositol 3-kinase (PI 3-kinase) in A549 human epithelial cells. Pre-treatment with the PI 3-kinase-specifi c inhibitors, wortmannin, and LY294002 effectively blocked BK-induced PI 3- kinase activity. Wortmannin and LY294002 also abolished BK-induced NF-kappa B activation, as did transient transfection with a dominant negative mutan t of the p85 subunit, BR-stimulated PI 3-kinase activity and NF-kappa B act ivation were sensitive to pertussis but not cholera toxin, suggesting that the B2 BK receptors transducing the response were coupled to G alpha i or G alpha o heterotrimeric G proteins. Tumor necrosis factor alpha (TNF alpha) also stimulated increased PI 3-kinase activity, however TNF alpha-stimulat ed NF-kappa B activation was not affected by the PI 3-kinase inhibitors or the p85 dominant negative mutant. These findings provide evidence that BK-i nduced NF-kappa B activation utilizes a signaling pathway that requires act ivity of both RhoA and PI 3-kinase and is distinct from the signaling pathw ay utilized by TNF alpha, Furthermore, we show that the p85 regulatory subu nit is required for activation of PI 3-kinase activity by this G protein-co upled receptor.