Dual Pathways for Nuclear Factor &kgr;B Activation by Angiotensin II in Vascular Smooth Muscle: Phosphorylation of p65 by I&kgr;B Kinase and Ribosomal Kinase

Activation of nuclear factor (NF)-&kgr;B by angiotensin II (Ang II) plays an essential role in stimulating expression of vascular adhesion molecules, which are essential for vascular inflammation. We report that Ang II activates NF-&kgr;B by phosphorylating its p65 subunit via a pathway mediated partially by ribosomal S6 kinase (RSK). In investigating other pathway(s) that may be involved, we found that the ability of Ang II to activate NF-&kgr;B in mouse embryonic fibroblast is suppressed (≈70%) either by deletion of I&kgr;B Kinase (IKK) or by inhibiting or knocking down IKK in vascular smooth muscle cells using a dominant-negative IKK adenovirus or small interference RNA to IKKβ. Thus, Ang II also stimulates NF-&kgr;B via IKK. In vitro, we found that Ang II stimulates IKK to phosphorylate myelin basic protein and the p65 subunit of NF-&kgr;B. The mechanism by which Ang II activates IKK is to increase phosphorylation of IKKβ in its activation loop (Ser181) rather than I&kgr;B phosphorylation. Inhibiting both the RSK and IKK pathways completely blocks the Ang II–induced p65 phosphorylation and NF-&kgr;B activation. These 2 pathways are independent: inhibiting IKK does not block Ang II–induced phosphorylation of RSK, whereas inhibiting mitogen-activated protein kinase 1 does not affect phosphorylation of IKK. Finally, we found that Ang II can induce expression of vascular adhesion molecules by 2 pathways; both IKK and RSK lead to phosphorylation of the p65 subunit of NF-&kgr;B to increase vascular cell adhesion molecule-1 transcription. The 2 pathways are functionally important because inhibiting IKK and RSK in vascular smooth muscle cells blocks Ang II–induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to limit vascular inflammation.