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

Abstract

ABSTRACT Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor κB (NF-κB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994). Recently, we reported a biphasic pattern of NF-κB 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-κB 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-κB 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-κB 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 ATPγS, 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 IκBα. This lack of IκBα involvement was supported by the finding that R. rickettsii can induce NF-κB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IκBα, rendering NF-κB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-κB 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.