The avb3 integrin plays an essential role in angiogenesis by inhibiting endothelial cell apoptosis. The mechanisms involved in conferring endothelial survival have not been well defined. Recently, we have shown that avb3 ligation on rat aortic endothelial cells (RAECs) specifically activates the transcription factor NF-kB (Scatena et al, 1998). The protective effect of avb3 ligands was abolished by inhibiting NF-kB nuclear translocation. To define the signaling mediators involved in avb3–mediated NF-kB activation we transiently transfected RAECs with an NF-kB response reporter construct in combination with wild type (WT) and dominant negative (DN) constructs encoding known upstream mediators of NF-kB signaling: inhibitor of kappa B kinase alpha (IKKa), inhibitor of kappa B kinase beta (IKKb), and NF-kB-inducing kinase (NIK). Cells plated on the avb3 ligand, osteopontin (OPN), showed approximately a 4-fold induction of NF-kB activity when compared to control cells plated poly-D-lysine (PDL). OPN-mediated NF-kB activity was increased upon NIK WT transfection and blocked following NIK DN transfection. IKKb appeared to be the main IkB-kinase, since OPN-induced NF-kB activity was enhanced in response to IKKb WT transfection, and blocked in response to IKKb DN. Finally, IkB-a phosphorylation, was observed in cells plated on OPN but not in cells plated on PDL as measured by Western blot. These studies suggest that avb3 ligation stimulates IKKb activity via NIK, and leads to phosphorylation of IkBa and subsequent NF-kB activation. Future studies are aimed at gathering biochemical evidence for NIK/IKK complex phosphorylation and functional activation, as well as defining the upstream mediators of NIK activation in response to OPN-avb3 ligation