Establishment of Lipopolysaccharide-dependent Nuclear Factor κB Activation in a Cell-free System (*)

Nuclear factor κB (NF-κB), consisting of p50 and p65, is bound to a cytoplasmic retention protein, IκB, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-κB from IκB and the nuclear translocation of NF-κB, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor α. In order to elucidate the precise mechanism of NF-κB activation, we have established lipopolysaccharide (LPS)-dependent NF-κB activation in a cell-free system using plasma membrane-enriched, cytosol, and nuclear fractions extracted from a human monocytic cell line, THP-1, by disruption with sonication followed by a differential centrifugation. The combination of plasma membrane-enriched fraction and cytosol was sufficient to activate NF-κB in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-κB to the IL-8 gene κB site on an electrophoretic mobility shift assay. LPS-dependent NF-κB activation was inhibited by protein kinase inhibitors, such as staurosporine, herbimycin A, tyrphostin, and genistein, but not mitogen-activated protein kinase substrate, cGMP-dependent protein kinase, cAMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II inhibitory peptides, suggesting that staurosporine-sensitive kinase(s) as well as tyrosine kinase(s) are involved in LPS-mediated NF-κB activation. In addition, LPS induced the phosphorylation of IκB-α, starting at 5 min after the stimulation in a cell-free system. Moreover, the phosphorylation was inhibited by herbimycin A and tyrphostin, but not staurosporine, suggesting that these protein kinase inhibitors act at distinct steps of signal transmission. Establishment of ligand-dependent activation of NF-κB in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-κB activation.