Selective inhibition of inhibitory kappa B kinase-beta abrogates induction of nitric oxide synthase in lipopolysaccharide-stimulated rat aortic smooth muscle cells.

In this study, we utilised a number of adenoviral constructs in order to examine the role of intermediates of the NF-kappaB pathway in the regulation of inducible nitric oxide synthase (iNOS) induction in rat aortic smooth muscle cells (RASMCs). Lipopolysaccharide (LPS) stimulated a significant increase in iNOS induction and NF-kappaB DNA binding. These parameters were substantially reduced by overexpression of a wild-type Ikappa-Balpha adenoviral construct (Ad.Ikappa-Balpha), confirming a role for NF-kappaB in iNOS induction. Infection with a dominant-negative IKKalpha adenoviral construct (Ad.IKKalpha+/-) did not significantly affect iNOS induction, NF-kappaB DNA binding or Ikappa-Balpha loss. Infection of RASMCs with adenovirus encoding a dominant-negative IKKbeta (Ad.IKKbeta+/-) essentially abolished iNOS induction and activation of the NF-kappaB pathway. Pretreatment of RASMCs with a novel specific inhibitor of IKKbeta, SC-514, significantly reduced iNOS induction, NF-kappaB DNA binding and I-kappaBalpha loss in a concentration-dependent manner. In both RASMCs and human umbilical vein endothelial cells (HUVECs), infection with Ad.IKKbeta+/- also inhibited COX-2 expression in response to LPS. However, Ad.IKKalpha+/- was again without effect. These data suggest that IKKbeta plays a predominant, selective role in the regulation of NF-kappaB-dependent induction of iNOS in RASMCs.