ABSTRACT
Tumour necrosis factor (TNF) is an inflammatory cytokine possessing a unique property: it can induce cells to undergo apoptosis. The sensitivity of different cell types to TNF-induced apoptosis can vary dramatically, but most cells become very sensitive upon simultaneous treatment with inhibitors of protein synthesis. It has been suggested therefore that a gene, or set of genes, is induced upon TNF receptor activation that downregulates the apoptosis signal. Recent results have shown that NF-kappa B, a transcription factor activated upon TNF signalling, is at least partly responsible for this effect. These findings have broadened the role of NF-kappa B from that of a regulator of immune and inflammatory responses to include an involvement in the regulation of apoptosis.
Subject(s)
Apoptosis/physiology , NF-kappa B/physiology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Apoptosis/drug effects , HumansABSTRACT
Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor kappaB (NF-kappaB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-kappaB 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-kappaB 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-kappaB 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-kappaB 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 ATPgammaS, 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 IkappaB alpha. This lack of IkappaB alpha involvement was supported by the finding that R. rickettsii can induce NF-kappaB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IkappaB alpha, rendering NF-kappaB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-kappaB 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.
Subject(s)
Cysteine Endopeptidases/physiology , Endothelium, Vascular/metabolism , I-kappa B Proteins , Multienzyme Complexes/physiology , NF-kappa B/metabolism , Rickettsia rickettsii/physiology , Cells, Cultured , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Endothelium, Vascular/cytology , Humans , NF-KappaB Inhibitor alpha , NF-kappa B/chemistry , Phosphorylation , Proteasome Endopeptidase Complex , Signal TransductionABSTRACT
Tumor necrosis factor alpha (TNF-alpha) signaling gives rise to a number of events, including activation of transcription factor NF-kappaB and programmed cell death (apoptosis). Previous studies of TNF-alpha signaling have suggested that these two events occur independently. The sensitivity and kinetics of TNF-alpha-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative IkappaBalpha (IkappaBalphaM). These findings suggest that a negative feedback mechanism results from TNF-alpha signaling in which NF-kappaB activation suppresses the signals for cell death.
Subject(s)
Apoptosis , I-kappa B Proteins , NF-kappa B/antagonists & inhibitors , NF-kappa B/physiology , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacology , Animals , Annexin A5/metabolism , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Feedback , Humans , Jurkat Cells , Mice , NF-KappaB Inhibitor alpha , Phosphatidylserines/metabolism , Transcription Factor RelAABSTRACT
Signal-induced degradation of I(kappa)B(alpha) via the ubiquitin-proteasome pathway requires phosphorylation on residues serine 32 and serine 36 followed by ubiquitination on lysines 21 and 22. We investigated the role of other regions of I(kappa)B(alpha) which may be involved in its degradation. Here we report that the carboxy-terminal PEST sequence is not required for I(kappa)B(alpha) signal-induced degradation. However, removal of the PEST sequence stabilizes free I(kappa)B(alpha) in unstimulated cells. We further report that a PEST deletion mutant does not associate well with NF-(kappa)B proteins but is degraded in response to signal. Therefore, we conclude that both association with NF-(kappa)B and a PEST sequence are not required for signal-induced I(kappa)B(alpha) degradation. Additionally, the PEST sequence may be required for constitutive turnover of free I(kappa)B(alpha).
