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 bound to a cytoplasmic retention protein, I kappa B, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-kappa B from I kappa B and the nuclear translocation of NF-kappa B, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NF-kappa B activation, we have established lipopolysaccharide (LPS)-dependent NF-kappa B 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-kappa B in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-kappa B to the IL-8 gene kappa B site on an electrophoretic mobility shift assay. LPS-dependent NF-kappa B 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-kappa B activation. In addition, LPS induced the phosphorylation of I kappa B-alpha, 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-kappa B in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappa B activation.
Ishikawa
Y
Y
Department of Pharmacology, Kanazawa University, Japan.
Mukaida
N
N
Kuno
K
K
Rice
N
N
Okamoto
S
S
Matsushima
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051841:Transcription Factor RelB; D014157:Transcription Factors
D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D014157:Transcription Factors; D051841:Transcription Factor RelB