Nuclear factor B (NF-B), consisting of p50 and p65, is bound to a cytoplasmic retention protein, IB, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-B from IB and the nuclear translocation of NF-B, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor . In order to elucidate the precise mechanism of NF-B activation, we have established lipopolysaccharide (LPS)-dependent NF-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-B in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-B to the IL-8 gene B site on an electrophoretic mobility shift assay. LPS-dependent NF-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-B activation. In addition, LPS induced the phosphorylation of IB-, 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-B in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-B activation.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Hasegawa, R. Imamura, K. Motani, T. Nishiuchi, N. Matsumoto, T. Kinoshita, and T. Suda Mechanism and Repertoire of ASC-Mediated Gene Expression J. Immunol., June 15, 2009; 182(12): 7655 - 7662. [Abstract] [Full Text] [PDF]

				J. Hisatsune, M. Nakayama, H. Isomoto, H. Kurazono, N. Mukaida, A. K. Mukhopadhyay, T. Azuma, Y. Yamaoka, J. Sap, E. Yamasaki, et al. Molecular Characterization of Helicobacter pylori VacA Induction of IL-8 in U937 Cells Reveals a Prominent Role for p38MAPK in Activating Transcription Factor-2, cAMP Response Element Binding Protein, and NF-{kappa}B Activation J. Immunol., April 1, 2008; 180(7): 5017 - 5027. [Abstract] [Full Text] [PDF]

				R. Imamura, K. Konaka, N. Matsumoto, M. Hasegawa, M. Fukui, N. Mukaida, T. Kinoshita, and T. Suda Fas Ligand Induces Cell-autonomous NF-{kappa}B Activation and Interleukin-8 Production by a Mechanism Distinct from That of Tumor Necrosis Factor-{alpha} J. Biol. Chem., November 5, 2004; 279(45): 46415 - 46423. [Abstract] [Full Text] [PDF]

				L. Ulloa, M. Ochani, H. Yang, M. Tanovic, D. Halperin, R. Yang, C. J. Czura, M. P. Fink, and K. J. Tracey Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation PNAS, September 17, 2002; 99(19): 12351 - 12356. [Abstract] [Full Text] [PDF]

				I. Feoktistov, A. E. Goldstein, S. Ryzhov, D. Zeng, L. Belardinelli, T. Voyno-Yasenetskaya, and I. Biaggioni Differential Expression of Adenosine Receptors in Human Endothelial Cells: Role of A2B Receptors in Angiogenic Factor Regulation Circ. Res., March 22, 2002; 90(5): 531 - 538. [Abstract] [Full Text] [PDF]

				M. Galdiero, M. Vitiello, E. Sanzari, M. D'Isanto, A. Tortora, A. Longanella, and S. Galdiero Porins from Salmonella enterica Serovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-{kappa}B through the Raf-1-Mitogen-Activated Protein Kinase Cascade Infect. Immun., February 1, 2002; 70(2): 558 - 568. [Abstract] [Full Text] [PDF]

				M. Yeh, N. Leitinger, R. de Martin, N. Onai, K. Matsushima, D. K. Vora, J. A. Berliner, and S. T. Reddy Increased Transcription of IL-8 in Endothelial Cells Is Differentially Regulated by TNF-{alpha} and Oxidized Phospholipids Arterioscler. Thromb. Vasc. Biol., October 1, 2001; 21(10): 1585 - 1591. [Abstract] [Full Text] [PDF]

				J. S. Friedland, D. Constantin, T. C. Shaw, and E. Stylianou Regulation of interleukin-8 gene expression after phagocytosis of zymosan by human monocytic cells J. Leukoc. Biol., September 1, 2001; 70(3): 447 - 454. [Abstract] [Full Text] [PDF]

				S. Hiraoka, Y. Miyazaki, S. Kitamura, M. Toyota, T. Kiyohara, Y. Shinomura, N. Mukaida, and Y. Matsuzawa Gastrin induces CXC chemokine expression in gastric epithelial cells through activation of NF-{kappa}B Am J Physiol Gastrointest Liver Physiol, September 1, 2001; 281(3): G735 - G742. [Abstract] [Full Text] [PDF]

				R. W. Ganster, B. S. Taylor, L. Shao, and D. A. Geller Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B PNAS, June 28, 2001; (2001) 151239498. [Abstract] [Full Text] [PDF]

				X. Zhang, J. M. Wang, W. H. Gong, N. Mukaida, and H. A. Young Differential Regulation of Chemokine Gene Expression by 15-Deoxy-{{Delta}}12,1412,14 Prostaglandin J2 J. Immunol., June 15, 2001; 166(12): 7104 - 7111. [Abstract] [Full Text] [PDF]

				P. Farmer and J. Pugin beta -Adrenergic agonists exert their "anti-inflammatory" effects in monocytic cells through the Ikappa B/NF-kappa B pathway Am J Physiol Lung Cell Mol Physiol, October 1, 2000; 279(4): L675 - L682. [Abstract] [Full Text] [PDF]

				G. W. Peet and J. Li Ikappa B Kinases alpha and beta Show a Random Sequential Kinetic Mechanism and Are Inhibited by Staurosporine and Quercetin J. Biol. Chem., November 12, 1999; 274(46): 32655 - 32661. [Abstract] [Full Text] [PDF]

				V. Faure, Y. Courtois, and O. Goureau Tyrosine kinase inhibitors and antioxidants modulate NF-kappa B and NOS-II induction in retinal epithelial cells Am J Physiol Cell Physiol, July 1, 1998; 275(1): C208 - C215. [Abstract] [Full Text] [PDF]

				K.-i. Kinugawa, T. Shimizu, A. Yao, O. Kohmoto, T. Serizawa, and T. Takahashi Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes Circ. Res., December 19, 1997; 81(6): 911 - 921. [Abstract] [Full Text]

				S. D. Savkovic, A. Koutsouris, and G. Hecht Activation of NF-kappa B in intestinal epithelial cells by enteropathogenic Escherichia coli Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1160 - C1167. [Abstract] [Full Text] [PDF]

				Y. Sonoda, T. Kasahara, Y. Yamaguchi, K. Kuno, K. Matsushima, and N. Mukaida Stimulation of Interleukin-8 Production by Okadaic Acid and Vanadate in a Human Promyelocyte Cell Line, an HL-60 Subline. POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE ON THE OKADAIC ACID-INDUCED NF-kappa B ACTIVATION J. Biol. Chem., June 13, 1997; 272(24): 15366 - 15372. [Abstract] [Full Text] [PDF]

				F. Meng and C. A. Lowell Lipopolysaccharide (LPS)-induced Macrophage Activation and Signal Transduction in the Absence of Src-Family Kinases Hck, Fgr, and Lyn J. Exp. Med., May 5, 1997; 185(9): 1661 - 1670. [Abstract] [Full Text] [PDF]

				K. Kuno, Y. Ishikawa, M. K. Ernst, M. Ogata, N. R. Rice, N. Mukaida, and K. Matsushima Identification of an IkappaBalpha-associated Protein Kinase in a Human Monocytic Cell Line and Determination of Its Phosphorylation Sites on IkappaBalpha J. Biol. Chem., November 17, 1995; 270(46): 27914 - 27919. [Abstract] [Full Text] [PDF]

				Z Chen, J Hagler, V J Palombella, F Melandri, D Scherer, D Ballard, and T Maniatis Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes & Dev., July 1, 1995; 9(13): 1586 - 1597. [Abstract] [PDF]

				R. W. Ganster, B. S. Taylor, L. Shao, and D. A. Geller Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B PNAS, July 17, 2001; 98(15): 8638 - 8643. [Abstract] [Full Text] [PDF]

				I. Feoktistov, A. E. Goldstein, S. Ryzhov, D. Zeng, L. Belardinelli, T. Voyno-Yasenetskaya, and I. Biaggioni Differential Expression of Adenosine Receptors in Human Endothelial Cells: Role of A2B Receptors in Angiogenic Factor Regulation Circ. Res., March 22, 2002; 90(5): 531 - 538. [Abstract] [Full Text] [PDF]