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Infect Immun, May 1998, p. 1827-1833, Vol. 66, No. 5
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Proteasome-Independent Activation of Nuclear Factor kappa B in Cytoplasmic Extracts from Human Endothelial Cells by Rickettsia rickettsii

Sanjeev K. Sahni,1,* Daniel J. Van Antwerp,2 Marina E. Eremeeva,3 David J. Silverman,3 Victor J. Marder,1,4 and Lee Ann Sporn1,4

Vascular Medicine Unit, Department of Medicine,1 and Department of Pathology and Laboratory Medicine,4 University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Laboratory of Genetics, Salk Institute, La Jolla, California 920372; and Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 212013

Received 8 October 1997/Returned for modification 24 November 1997/Accepted 26 January 1998

Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor kappa B (NF-kappa B) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-kappa B activation in cultured human umbilical vein endothelial cells consequent to infection with Rickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786-2791, 1997). In the present study, we describe activation of NF-kappa B in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of R. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-kappa B subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-kappa B pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPgamma S, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of Ikappa Balpha . This lack of Ikappa Balpha involvement was supported by the finding that R. rickettsii can induce NF-kappa B activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of Ikappa Balpha , rendering NF-kappa B inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-kappa B activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.

* Corresponding author. Mailing address: Vascular Medicine Unit, Department of Medicine, Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Phone: (716) 275-5186. Fax: (716) 473-4314. E-mail: ssahni{at}medicine.rochester.edu.

Infect Immun, May 1998, p. 1827-1833, Vol. 66, No. 5
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

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