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

The signaling mechanisms utilized by bradykinin (BK) to activate the transcription factor nuclear factor kappa B (NF-kappa B) are poorly defined. We previously demonstrated that BK-stimulated NF-kappa B activation requires the small GTPase RhoA, We present evidence that BK-induced NF-kappa B activation both activates and requires phosphatidylinositol 3-kinase (PI 3-kinase) in A549 human epithelial cells. Pre-treatment with the PI 3-kinase-specific 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 mutant of the p85 subunit, BR-stimulated PI 3-kinase activity and NF-kappa B activation 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-stimulated 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-induced NF-kappa B activation utilizes a signaling pathway that requires activity of both RhoA and PI 3-kinase and is distinct from the signaling pathway utilized by TNF alpha, Furthermore, we show that the p85 regulatory subunit is required for activation of PI 3-kinase activity by this G protein-coupled receptor.