Proteasome-independent activation of nuclear factor kappaB 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 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.

FIG. 1

EMSA of NF-κB activation in cytoplasmic extracts of endothelial cells. (A) Aliquots of cytoplasmic extracts (cyt) were treated with 1.0% NP-40 and 0.6% DOC (NP40/DOC) at 37°C for 30 min prior to assay of NF-κB activation with a double-stranded NF-κB oligonucleotide probe. Specificity of complex formation was confirmed by addition of a 10-fold molar excess of unlabeled oligonucleotide (+cold). Gel shift with nuclear extract from HeLa cells (5 μg of protein) was used as a positive control for DNA binding activity of NF-κB. (B) Cytoplasmic extracts (20-μl aliquots) were incubated alone (cyt) or in the presence of approximately 4 × 10⁵ PFU of partially purified R. rickettsii (cyt+RR) for 30 min prior to assay. Rickettsial preparations alone (RR; 4 × 10⁵ PFU) were assayed to control for the presence of contaminating, activated NF-κB. The relative positions of gel-shifted complexes p50-p65 and p50-p50 are shown; NS represents a nonspecific complex. Autoradiographic exposures were from 12 to 18 h with Kodak intensifying screens. Excess, unbound ³²P-labeled oligonucleotide migrated to the bottom of the gel and is indicated as free probe.

FIG. 2

Concentration (PFU) and time dependence of in vitro NF-κB activation in endothelial cell cytoplasmic extracts. (A) Aliquots of cytoplasmic extracts from unstimulated endothelial cells (cyt) were incubated at 37°C for 30 min with undiluted R. rickettsii (RR) along with R. rickettsii diluted 1:5, 1:20, and 1:100 (RR/5, RR/20, and RR/100, respectively), followed by EMSA. Lanes marked +cold indicate the presence of a 10-fold molar excess of unlabeled probe in the binding reaction mixture. Cytoplasmic extract was also treated with NP-40 and DOC as described for Fig. 1A. NS, nonspecific complex. (B) Endothelial cell cytoplasmic extracts were incubated with approximately 10⁵ PFU of partially purified R. rickettsii for 15, 30, 60, and 120 min, followed by EMSA. The autoradiogram of the gel was scanned as described in Materials and Methods, and band intensities were corrected by subtraction of baseline levels of NF-κB in cytoplasmic extracts and R. rickettsii preparations alone.

FIG. 3

Antibody supershift analysis of activated NF-κB species. Endothelial cell cytoplasmic extracts incubated with R. rickettsii undiluted (RR) and diluted 1:20 (RR/20) at 37°C for 30 min were subjected to gel supershift analysis using antibodies against the NF-κB subunits p50 and p65. Antibody (2 μg) was incubated with R. rickettsii-treated cytoplasmic extracts at 37°C for 20 min prior to the addition of radiolabeled oligonucleotide probe. Supershifted complexes on the gel are indicated by arrows, and the original locations of gel-shifted complexes are indicated by the arrowheads. NS, nonspecific complex. Lanes are labeled as for Fig. 2.

FIG. 4

Effect of proteasome inhibitor MG 132 on R. rickettsii-induced activation of NF-κB in cytoplasmic extracts and intact cells. (A) Endothelial cell cytoplasmic extracts were preincubated with MG 132 at 37°C for 30 min prior to incubation with R. rickettsii (cyt+RR+MG132) or with dimethyl sulfoxide (used as solvent) prior to addition of R. rickettsii (cyt+RR). EMSA was then performed to visualize activated NF-κB complexes. Treatment with MG 115 yielded similar results. NS, nonspecific complex. (B) Nuclear extracts were prepared from uninfected cultures (C), uninfected cultures treated with 50 μM MG 132 for 1 h (C+MG132), cultures infected with R. rickettsii for 3 h (RR), or cultures treated with MG 132 (25 and 50 μM) for 1 h prior to and incubation with R. rickettsii (RR+MG132); 5 μg of nuclear protein was used in each gel shift reaction. Similar inhibition was seen with MG 115 (not shown).

FIG. 5

ATP independence of R. rickettsii-induced NF-κB activation. Endothelial cell cytoplasmic extracts (cyt) were preincubated at 37°C for 30 min with ATP, ATPγS, and ATPase individually, at concentrations indicated, prior to incubation with R. rickettsii (RR) or buffer alone. The samples were analyzed by EMSA for comparison between the levels of activated NF-κB species.

FIG. 6

Western blot analysis for levels of IκBα and phospho-IκBα (Ser32) during in vitro activation of NF-κB by R. rickettsii. Lane 1, extract from HeLa cells (control for IκBα); lane 2, extract from TNF-α-treated HeLa cells (control for phospho-IκBα); lane 3, cytoplasmic extract alone from untreated endothelial cells; lane 4, cytoplasmic extract from endothelial cells to which R. rickettsii was added at 4°C and immediately frozen; lane 5, cytoplasmic extract from endothelial cells incubated with R. rickettsii at 37°C for 30 min to activate NF-κB; lane 6, cytoplasmic extract from endothelial cells incubated with R. rickettsii at 37°C for 30 min followed by removal of R. rickettsii by centrifugation prior to gel electrophoresis; lane 7, R. rickettsii preparation alone. Equal amounts of cytoplasmic protein were loaded on lanes 3 through 6. Relative positions of size markers (lane M) are shown at the left in kilodaltons.

FIG. 7

R. rickettsii-induced NF-κB activation in cytoplasmic extracts of T24 bladder carcinoma cells transfected with either empty LXSN vector (T24cyt) or IκBαM-containing vector (MutT24cyt). Cytoplasmic extracts (20-μl aliquots) were incubated alone or in the presence of 4 × 10⁵ PFU of partially purified R. rickettsii (+RR) for 30 min prior to assay. Preparation of R. rickettsii (RR) was also analyzed to control for the presence of any preexisting activated NF-κB species. NS, nonspecific complex.