Enhancement of lipopolysaccharide-stimulated JNK activity in rat aortic smooth muscle cells by pharmacological and adenovirus-mediated inhibition of inhibitory kappa B kinase signalling

Abstract

1. In rat aortic smooth muscle cells (RASMCs), the putative nuclear factor kappa B (NFκB) inhibitor Pyrrolidine dithiocarbamate (PDTC) was found to inhibit lipopolysaccharide (LPS)-stimulated NFκB DNA-binding. However, further investigation identified the site of inhibition as being at, or upstream of, the inhibitory kappa B kinases (IKKs) as their kinase activity was substantially reduced. 2. In addition, PDTC potentiated LPS-stimulated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAP kinase) and MAP kinase-activated protein kinase-2 activity (the downstream target of p38 MAP kinase). 3. Another inhibitor of NFκB signalling, the serine protease inhibitor Nαp-tosyl-L-lysine chloromethylketone (TLCK), also inhibited LPS-stimulated IKK activity and potentiated JNK activity in response to LPS, suggesting that cross-talk may occur between the NFκB and stress-activated protein kinase pathways at the level of IKK or at a common point upstream. 4. Infection of RASMCs with an adenovirus encoding either inhibitory kappa Bα or a dominant-negative IKKβ potentiated LPS-stimulated JNK activity. 5. These studies therefore suggest that the loss of NFκB DNA-binding and resultant transcriptional activity, rather than the loss of IKK activity, is sufficient to cause an increase in JNK activity. This shows that either pharmacological or molecular inhibition of NFκB DNA-binding enhances JNK activation in vascular smooth muscle cells, an effect that may contribute to the pathophysiological effects of LPS.