Activation of NF-κB via the IκB kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells

Activation of the transcription factor NF-kappaB is necessary for full expression of tumor necrosis factor alpha (TNF-alpha)-inducible endothelial chemokines and adhesion molecules. However, a detailed analysis regarding contribution of the different NF-kappaB upstream components to endothelial activation has not been performed yet. We employed a retroviral infection approach to stably express transdominant (TD) mutants of I kappaB alpha, I kappaB beta, or I kappaB epsilon and dominant negative (dn) versions of I kappaB kinases (IKK) 1 or 2 as well as a constitutively active version of IKK2 in human endothelial cells. TD I kappaB alpha, I kappaB beta, and I kappaB epsilon were not degraded upon TNF-alpha exposure, and each prevented NF-kappaB activation. These TD I kappaB mutants almost completely inhibited the induction of monocyte chemoattractant protein-1, interleukin-8, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression by TNF-alpha, whereas interferon-gamma -mediated up-regulation of intercellular adhesion molecule-1 and HLA-DR was not affected. Expression of dn IKK2 completely blocked TNF-alpha -induced up-regulation, whereas dn IKK1 showed a partial inhibition of expression of these molecules. Importantly, expression of constitutively active IKK2 was sufficient to drive full expression of all chemokines and adhesion molecules in the absence of cytokine. We conclude that the IKK/I kappaB/NF-kappaB pathway is crucial and sufficient for proinflammatory activation of endothelium.