Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii

Sanjeev Sahni, Daniel J. Van Antwerp, Marina E. Eremeeva, David J. Silverman, Victor J. Marder, Lee Ann Sporn

Research output: Contribution to journalArticle

43 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1827-1833
Number of pages7
JournalInfection and Immunity
Volume66
Issue number5
StatePublished - May 1998
Externally publishedYes

Fingerprint

Rickettsia rickettsii
Proteasome Endopeptidase Complex
Endothelial Cells
Adenosine Triphosphate
Rocky Mountain Spotted Fever
Phosphorylation
Proteasome Inhibitors
Cell-Free System
Human Umbilical Vein Endothelial Cells
Eukaryotic Cells
Cell Extracts
Gram-Negative Bacteria
Aldehydes
Serine
Adenosine Triphosphatases
Immune Sera
Signal Transduction
Urinary Bladder
Hydrolysis
Transcription Factors

ASJC Scopus subject areas

  • Immunology

Cite this

Sahni, S., Van Antwerp, D. J., Eremeeva, M. E., Silverman, D. J., Marder, V. J., & Sporn, L. A. (1998). Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii. Infection and Immunity, 66(5), 1827-1833.

Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii. / Sahni, Sanjeev; Van Antwerp, Daniel J.; Eremeeva, Marina E.; Silverman, David J.; Marder, Victor J.; Sporn, Lee Ann.

In: Infection and Immunity, Vol. 66, No. 5, 05.1998, p. 1827-1833.

Research output: Contribution to journalArticle

Sahni, S, Van Antwerp, DJ, Eremeeva, ME, Silverman, DJ, Marder, VJ & Sporn, LA 1998, 'Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii', Infection and Immunity, vol. 66, no. 5, pp. 1827-1833.
Sahni, Sanjeev ; Van Antwerp, Daniel J. ; Eremeeva, Marina E. ; Silverman, David J. ; Marder, Victor J. ; Sporn, Lee Ann. / Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii. In: Infection and Immunity. 1998 ; Vol. 66, No. 5. pp. 1827-1833.
@article{971ebfb43f8643548fa4708990de631c,
title = "Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii",
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.",
author = "Sanjeev Sahni and {Van Antwerp}, {Daniel J.} and Eremeeva, {Marina E.} and Silverman, {David J.} and Marder, {Victor J.} and Sporn, {Lee Ann}",
year = "1998",
month = "5",
language = "English (US)",
volume = "66",
pages = "1827--1833",
journal = "Infection and Immunity",
issn = "0019-9567",
publisher = "American Society for Microbiology",
number = "5",

}

TY - JOUR

T1 - Proteasome-independent activation of nuclear factor κB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii

AU - Sahni, Sanjeev

AU - Van Antwerp, Daniel J.

AU - Eremeeva, Marina E.

AU - Silverman, David J.

AU - Marder, Victor J.

AU - Sporn, Lee Ann

PY - 1998/5

Y1 - 1998/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0031924778&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031924778&partnerID=8YFLogxK

M3 - Article

VL - 66

SP - 1827

EP - 1833

JO - Infection and Immunity

JF - Infection and Immunity

SN - 0019-9567

IS - 5

ER -