The Journal of Immunology, Vol 153, Issue 10 4713-4720, Copyright © 1994 by American Association of Immunologists

ARTICLES

Murine inhibitory protein-kappa B alpha negatively regulates kappa B- dependent transcription in lipopolysaccharide-stimulated RAW 264.7 macrophages

JM Tebo, W Chaoqun, Y Ohmori and TA Hamilton
Department of Immunology, Cleveland Clinic Foundation, Ohio 44195.

The potential role of the inhibitory protein (I)-kappa B alpha gene in control of LPS-dependent transcription has been investigated in the murine macrophage cell line RAW 264.7. LPS-induced transcription in macrophages is believed to involve activation of members of the Rel homology family of transcription factors, and may be negatively regulated by cytoplasmic inhibitor proteins collectively termed I-kappa Bs. To evaluate the role of I-kappa Bs in LPS-stimulated macrophages, murine I-kappa B alpha (ml-kappa B alpha) has been expressed as a glutathione-S-transferase (GST) fusion protein and examined for its ability to control kappa B binding activities in nuclear extracts from LPS-treated RAW 264.7 macrophages. ml-kappa B alpha-GST inhibited LPS- induced kappa B binding activity from RAW 264.7 cells in a phosphorylation-dependent fashion, but did not affect IFN-alpha-induced IFN stimulus response element binding. Recombinant I-kappa B alpha inhibited kappa B motif binding by nuclear factor-kappa B1, RelA, and c- Rel as indicated by studies using UV radiation-induced covalent cross- linking to a bromodeoxyuridine-substituted kappa B oligonucleotide. Transfection of macrophages with an expression vector encoding ml-kappa B alpha inhibited LPS-stimulated transcription driven by a 243-bp promoter sequence obtained from the 5' flanking region of the murine IP- 10 gene. This promoter sequence contains two kappa B motifs that have been shown to be critical to LPS-dependent reporter gene transcription. The kappa B sites seem to be the specific target of I-kappa B alpha function, as reporter gene transcription driven by these motifs in the context of a heterologous thymidine-kinase promoter (TK) also was inhibited by co-transfection with ml-kappa B alpha. These observations indicate that ml-kappa B alpha is capable of controlling kappa B- dependent transcription in LPS-stimulated murine macrophages.

This article has been cited by other articles:

				J. Hong, S. Sang, H.-J. Park, S. J. Kwon, N. Suh, M.-T. Huang, C.-T. Ho, and C. S. Yang Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives Carcinogenesis, February 1, 2006; 27(2): 278 - 286. [Abstract] [Full Text] [PDF]

				S. O. Kim, K. Ono, P. S. Tobias, and J. Han Orphan Nuclear Receptor Nur77 Is Involved in Caspase-independent Macrophage Cell Death J. Exp. Med., June 2, 2003; 197(11): 1441 - 1452. [Abstract] [Full Text] [PDF]

				J. Fierer, M. A. Swancutt, D. Heumann, and D. Golenbock The Role of Lipopolysaccharide Binding Protein in Resistance to Salmonella Infections in Mice J. Immunol., June 15, 2002; 168(12): 6396 - 6403. [Abstract] [Full Text] [PDF]

				J. J. Watters, J. A. Sommer, Z. A. Pfeiffer, U. Prabhu, A. N. Guerra, and P. J. Bertics A Differential Role for the Mitogen-activated Protein Kinases in Lipopolysaccharide Signaling. THE MEK/ERK PATHWAY IS NOT ESSENTIAL FOR NITRIC OXIDE AND INTERLEUKIN 1beta PRODUCTION J. Biol. Chem., March 8, 2002; 277(11): 9077 - 9087. [Abstract] [Full Text] [PDF]

				S. O. Kim, K. Ono, and J. Han Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1095 - L1105. [Abstract] [Full Text] [PDF]

				D. J. Lee, D. Cox, J. Li, and S. Greenberg Rac1 and Cdc42 Are Required for Phagocytosis, but Not NF-kappa B-dependent Gene Expression, in Macrophages Challenged with Pseudomonas aeruginosa J. Biol. Chem., January 7, 2000; 275(1): 141 - 146. [Abstract] [Full Text] [PDF]

				J. Garcia, B. Lemercier, S. Roman-Roman, and G. Rawadi A Mycoplasma fermentans-derived Synthetic Lipopeptide Induces AP-1 and NF-kappa B Activity and Cytokine Secretion in Macrophages via the Activation of Mitogen-activated Protein Kinase Pathways J. Biol. Chem., December 18, 1998; 273(51): 34391 - 34398. [Abstract] [Full Text] [PDF]