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

Abstract

The signaling mechanisms utilized by bradykinin (BK) to activate the transcription factor nuclear factor κB (NF-κB) are poorly defined. We previously demonstrated that BK-stimulated NF-κB activation requires the small GTPase RhoA. We present evidence that BK-induced NF-κB 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-κB activation, as did transient transfection with a dominant negative mutant of the p85 subunit. BK-stimulated PI 3-kinase activity and NF-κB activation were sensitive to pertussis but not cholera toxin, suggesting that the B2 BK receptors transducing the response were coupled to Gαi or Gαo heterotrimeric G proteins. Tumor necrosis factor α (TNFα) also stimulated increased PI 3-kinase activity, however TNFα-stimulated NF-κB activation was not affected by the PI 3-kinase inhibitors or the p85 dominant negative mutant. These findings provide evidence that BK-induced NF-κB activation utilizes a signaling pathway that requires activity of both RhoA and PI 3- kinase and is distinct from the signaling pathway utilized by TNFα. Furthermore, we show that the p85 regulatory subunit is required for activation of PI 3-kinase activity by this G protein-coupled receptor.