RESUMO
IkappaB kinase (IKK) beta is essential for inflammatory cytokine-induced activation of nuclear factor kappaB (NF-kappaB). NF-kappaB plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKKbeta inhibitor N-(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-2-methylnicotinamide (ML120B), a beta-carboline derivative, on NF-kappaB signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKKbeta with an IC50 of 60 nM when evaluated in an IkappaBalpha kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentration-dependently inhibits tumor necrosis factor alpha (TNFalpha)-stimulated NF-kappaB signaling via inhibition of IkappaBalpha phosphorylation, degradation, and NF-kappaB translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNFalpha- or interleukin (IL)-1beta-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKKbeta-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide- or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKKbeta-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1beta-induced matrix metalloproteinase production with an IC50 of approximately 1 microM. ML120B also blocked IL-1beta-induced prostaglandin E2 production. In summary, ML120B blocked numerous NF-kappaB-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKKbeta inhibitors as anti-inflammatory agents for the treatment of RA.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células do Tecido Conjuntivo , Inibidores Enzimáticos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Condrócitos/imunologia , Células do Tecido Conjuntivo/efeitos dos fármacos , Células do Tecido Conjuntivo/enzimologia , Células do Tecido Conjuntivo/imunologia , Citocinas/imunologia , Dinoprostona/imunologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/imunologia , Células HeLa , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Mastócitos/imunologia , Estrutura Molecular , NF-kappa B/imunologia , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/citologiaRESUMO
Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IkappaB kinase beta (IKKbeta) in mucus synthesis in vitro and in vivo. Tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-alpha stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein. TNF-alpha stimulation induced IKKbeta-dependent p65 nuclear translocation, mucus synthesis, and production of cytokines from epithelial cells. TNF-alpha, but not transforming growth factor-alpha, induced mucus production dependent on IKKbeta-mediated NF-kappaB activation. In vivo, TNF-alpha induced NF-kappaB as determined by whole mouse body bioluminescence. This activation was localized to the epithelium as revealed by LacZ staining in NF-kappaB-LacZ transgenic mice. TNF-alpha-induced mucus production in vivo could also be inhibited by administration into the epithelium of an IKKbeta dominant negative adenovirus. Taken together, our results demonstrated the important role of IKKbeta in TNF-alpha-mediated mucus production in airway epithelium in vitro and in vivo.