ESTABLISHMENT OF LIPOPOLYSACCHARIDE-DEPENDENT NUCLEAR FACTOR KAPPA-B ACTIVATION IN A CELL-FREE SYSTEM

Nuclear factor kappa B (NF-kappa B), consisting of p50 and p65, is bou nd to a cytoplasmic retention protein, I kappa B, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli in duces the dissociation of NF-kappa B from I kappa B and the nuclear tr anslocation of NF-kappa B, thereby activating several genes involved i n inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NE-kappa B activation, we have established Lipopolysaccharide (LPS)-de pendent NF-kappa B activation in a cell-free system using plasma membr ane-enriched, cytosol, and nuclear fractions extracted from a human mo nocytic cell Line, THP-1, by disruption with sonication followed by a differential centrifugation. The combination of plasma membrane enrich ed fraction and cytosol was sufficient to activate NF-kappa B in a LPS /CD14-dependent manner only in the presence of ATP as judged by the bi nding of NF-kappa B to the IL-8 gene kappa B site on an electrophoreti c mobility shift assay. LPS-dependent NP-kappa B activation was inhibi ted by protein kinase inhibitors, such as staurosporine, herbimycin A, tyrphostin, and genistein, but not mitogen-activated protein kinase s ubstrate, cGMP-dependent protein kinase, cAMP-dependent protein kinase , protein kinase C, and calmodulin-dependent protein kinase II inhibit ory peptides, suggesting that staurosporine-sensitive kinase(s) as wel l as tyrosine kinase(s) are involved in LPS-mediated NF-kappa B activa tion. In addition, LPS induced the phosphorylation of I kappa B-alpha, starting at 5 min after the stimulation in a cell-free system. Moreov er, 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-kappa B in a cell-free system will facilit ate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappa B activation.