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NF-B Activation during Rickettsia rickettsii Infection of Endothelial Cells Involves the Activation of Catalytic IB Kinases IKK and IKKß and Phosphorylation-Proteolysis of the Inhibitor Protein IB

Dawn R. Clifton,^1, Elena Rydkina,² Robert S. Freeman,³ and Sanjeev K. Sahni^2*

Department of Environmental Medicine,¹ Hematology-Oncology Unit, Department of Medicine,² Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York³

Received 22 June 2004/ Returned for modification 27 July 2004/ Accepted 22 September 2004

	ABSTRACT

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Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-B (NF-B) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-B subunits, p65 and p50, and IB proteins, IB and IBß. The transcript and protein levels of p50, p65, and IBß remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IB at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IB mRNA. The level of IB mRNA gradually returned toward baseline, whereas that of total IB protein remained lower than the corresponding controls. The activities of IKK and IKKß, the catalytic subunits of IB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-B response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKK and IKKß, leading to attenuation of nuclear translocation of NF-B. Also, increased activity of IKK was evident later during the infection, coinciding with the late phase of NF-B activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-B activation. Since NF-B is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

	INTRODUCTION

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Rocky Mountain spotted fever, caused by the obligate intracellular bacterial pathogen Rickettsia rickettsii, is one of the most frequently reported and severe rickettsial diseases in the United States. R. rickettsii primarily invades vascular endothelial cells (ECs) in humans, replicates predominantly within the cytoplasm, and utilizes actin polymerization-based directional motility for intracellular movements and intercellular spread (19, 42). The vascular endothelium is a multifunctional endocrine and paracrine organ involved in the modulation of blood flow and vessel tone, coagulation, and regulation of immune and inflammatory responses. In general, an intricate relationship between activation of immune responses and modulation of coagulation properties, usually followed by dysregulation of hemostatic mechanisms, is a hallmark feature of infectious diseases affecting the endothelium (2, 45). A majority of pathological sequelae associated with spotted fever group rickettsioses are attributed to damage of ECs affecting these functions in severe cases of infection (41, 42). ECs not only participate in the uptake of viable Rickettsia organisms attached to the cell surface through "induced phagocytosis," resulting in internalization, but actively respond to infection by adjusting the expression of mediators with important physiological functions such as adhesion molecules, cytokines, chemokines, and regulatory components of the coagulation cascade (8, 40). Many of these genes are regulated by the nuclear factor-B (NF-B)/Rel family of transcription factors, known to play an important role in immediate-early pathogenic responses (1, 2, 12).

The prototypical NF-B complex is a heterodimer of RelA (p65) and p50 (NFKB1) subunits; other members of the Rel family include c-Rel, RelB, and p52 (NFKB2). In resting cells, NF-B is sequestered in the cytoplasm in its latent form through association with inhibitory proteins termed IB (IB, IBß, IB, IB, p105, and p100). Upon appropriate cell stimulation, IB and IBß are rapidly phosphorylated on specific amino-terminal serines, signaling for ubiquitination and degradation by the 26S proteasome (12, 46). This results in the exposure of a nuclear localization sequence (NLS) and DNA-binding domains, allowing NF-B to enter the nucleus and stimulate the transcription of target genes. One such target is IB itself, the resynthesis of which completes an autoregulatory loop to ensure that the activation of NF-B is turned off in a timely manner (12).

Despite sharing many common structural and functional properties (44), IB and IBß exhibit significant differences in their affinity for NF-B dimers (35). A recent study further suggests unique injury-context-specific roles for IB and IBß in NF-B-mediated cellular responses (9). Some agonists cause a transient activation of NF-B, e.g., tumor necrosis factor alpha (TNF-) and phorbol ester, inducing the rapid degradation and resynthesis of IB. However, other signals such as lipopolysaccharide (LPS) and interleukin-1 (IL-1) also cause the slower degradation of IBß (39). Newly synthesized IBß in a hypophosphorylated state is capable of binding to NF-B without masking the nuclear localization sequence, effectively preventing sequestration and subsequent inactivation of NF-B by newly synthesized IB, resulting in persistent NF-B activation (38). Thus, these differences are specific to variations in the phosphorylation state of IBß in response to a subset of inducers. A critical advance in the understanding of IB phosphorylation was the discovery of a multisubunit, high-molecular-weight complex termed IB kinase (IKK), which phosphorylates both IB and IBß. IKK contains two catalytically active subunits, IKK and IKKß (also referred to as IKK-1 and IKK-2), which form homo- and heterodimers through leucine zipper domains that bind to a third protein IKK (also called NEMO [for NF-B essential modulator]). Although IKK is a regulatory subunit with no catalytic activity, it is indispensable for coupling the IKK complex to upstream activators (17, 46).

Our laboratory has shown that activation of NF-B plays a crucial role during R. rickettsii infection of ECs by controlling the expression of several procoagulant and proinflammatory genes (34) and by exerting its antiapoptotic functions to protect the host cells from apoptotic death (3, 22). Interestingly, the kinetics of R. rickettsii-induced NF-B activation in ECs follows a biphasic pattern characterized by an early transient phase peaking at about 3 h postinfection (hpi) and a second sustained phase occurring between 18 and 24 h (37). In the present study, we describe the expression of the primary NF-B proteins p65 and p50, the phosphorylation and proteolysis of IB proteins, and the activation of catalytic components of IKK signalosome during R. rickettsii infection of ECs. The effect of specific IKK inhibitors on R. rickettsii-induced NF-B activation was also investigated.

	MATERIALS AND METHODS

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Cell culture, infection, and inactivation of Rickettsia organisms. All experiments were performed with human umbilical vein ECs cultures established as described previously (3, 37). ECs, seeded at a dilution to achieve 80 to 90% confluence after 3 to 4 days, were routinely used after a second passage in culture. Cultures were infected with the Sheila Smith strain of Rickettsia rickettsii by using a seed stock (1 x 10⁷ to 5 x 10⁷ PFU/ml) prepared from infected Vero cells (37). Unless stated otherwise, EC monolayers were infected with ca. 6 x 10⁴ PFU of viable rickettsia organisms for every square centimeter of culture area. As reported in our earlier studies, this protocol resulted in infection of ca. 75% of total cell population with two to three rickettsiae per cell at 3 h and 80% cells with three to four rickettsiae at 6 h (37). For inactivation, aliquots of rickettsial preparations were subjected to either heat treatment or fixation with formaldehyde (3.7% [vol/vol]) (30). Formaldehyde was removed by three washes with K36 buffer (0.1 M KCl, 0.15 M NaCl, 0.05 M potassium phosphate buffer [pH 7.0]) and centrifugation at 10,000 x g for 10 min.

RNA isolation and Northern blot analysis. Total RNA was isolated from uninfected and infected ECs at 3, 7, 14, and 21 hpi by using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's instructions. Northern blot analysis was performed essentially as described previously (14). Briefly, 20 µg of total RNA per condition was denatured in a glyoxal-dimethyl sulfoxide mix and then separated by electrophoresis in 1.4% agarose gels with 10 mM sodium phosphate buffer (pH 7.0) with recirculation. RNA was transblotted to Zeta-probe membrane (Bio-Rad Laboratories, Hercules, Calif.) in 0.5x TAE buffer (Tris-acetate, EDTA). The membrane was air dried, and RNA was fixed by baking at 80°C in vacuo for 1.5 h. Blots were prehybridized in 0.5 M Na₂HPO₄ (pH 7.2), 1 mM EDTA, and 7% sodium dodecyl sulfate (SDS) for at least 1 h. For detection of p65 and p105/p50, plasmids containing murine p65 and human p105/p50 cDNA (kindly provided by Edward Schwarz and Albert Baldwin, Jr., respectively) were used as described previously (48, 49). Restriction endonuclease digestion was performed on plasmids containing human IB and murine IBß (gifts from Edward Schwarz) to obtain the corresponding probes of 1,100 and 960 bp, respectively. Inserts were isolated by agarose gel electrophoresis, followed by purification by using Sephaglas BP kit (Amersham Biosciences, Piscataway, N.J.) according to the manufacturer's instructions. Prior to hybridization, the probes were radioactively labeled with [³²P]dCTP (Perkin-Elmer Life and Analytical Sciences, Boston, Mass.) by using the random primers DNA labeling system according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). The labeled probes were denatured by boiling, added to the prehybridization buffer, and incubated with the blots overnight. Blots were washed for 1 h in wash solution 1 (40 mM Na₂HPO₄ [pH 7.2], 1 mM EDTA, 5% SDS) and then three 30-min washes in wash solution 2 (40 mM Na₂HPO₄ [pH 7.2], 1 mM EDTA, 1% SDS). The temperatures used during hybridization and washing procedures for the murine and human probes were 58 and 65°C, respectively. For detection of signal, blots were exposed to Kodak Biomax MS film. Blots were stripped by repeating the wash solution protocol above except at 95°C. A human glyceraldehyde-3-phosphate dehydrogenase probe (GAPDH; 1,000 bp; provided by P. J. Simpson-Haidaris) was used to control for variations in sample loading and normalization of densitometric data.

Western blot analysis. At different times postinfection, protein lysates were prepared by scraping cells into 1x Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 2% [wt/vol] SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% [wt/vol] bromophenol blue) (25) and sonicated them for 10 s on ice to shear DNA. Equal volumes of total lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane. The primary antibodies for NF-B proteins were polyclonal rabbit anti-p50 and anti-p65 (Chemicon, Temecula, Calif.). Polyclonal anti-phospho-IB specifically detected phosphorylation at Ser-32, whereas a second polyclonal antibody detected total IB independent of N-terminal phosphorylation (Cell Signaling Technology, Beverly, Mass.). For detection of IBß proteins, an antibody recognizing an amino-terminal sequence common to both IBß₁ and IBß₂ isoforms was used (Santa Cruz Biotechnology, Santa Cruz, Calif.). A monoclonal murine anti-tubulin antibody (Amersham Biosciences) was used to normalize for sample loading. Blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, followed by Renaissance chemiluminescence reagent (Perkin-Elmer Life and Analytical Sciences) and subsequently exposed to ECL Hyperfilm (Amersham Biosciences).

IP of IKK/IKKß and measurement of kinase activity. For immunoprecipitation-kinase (IP-kinase) assays, cellular protein extracts were prepared in a buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, Mo.) as described previously (5), and the protein concentration was determined by Bradford assay. Equal amounts of sample protein (20 to 50 µg) were subjected to IP with either anti-IKK or anti-IKKß (Santa Cruz Biotechnology) or normal mouse immunoglobulin G (IgG) as a negative control. Typically, 2 µg of IKK or 3 µg of IKKß antibody was mixed with the cell lysates and incubated on a rocker platform for at least 2 h at 4°C. Protein A-Sepharose beads (10 µl) were then added, and the samples were incubated for an additional hour with gentle rotation. The protein A-Sepharose immunopellets were sedimented by centrifugation at 2,000 rpm for 2 min and washed twice with the IP buffer (20 mM HEPES-NaOH [pH 7.6], 40 mM sodium ß-glycerophosphate, 20 mM sodium fluoride, 1 mM sodium orthovanadate [activated], 20 mM p-nitrophenyl phosphate, 1 mM dithiothreitol, 0.1% [vol/vol] NP-40, 1 mM phenylmethylsulfonyl fluoride, and 10 µg of protease inhibitor cocktail/ml) supplemented with 0.4 M sodium chloride. The immune complexes for IKK were then washed with IP buffer containing 0.4 M sodium chloride and 2 M urea. After equilibration in a kinase reaction buffer (20 mM HEPES-NaOH [pH 7.6], 20 mM sodium ß-glycerophosphate, 10 mM magnesium chloride, 0.1 mM sodium orthovanadate [activated], 10 mM p-nitrophenyl phosphate, 50 mM sodium chloride, 2 mM dithiothreitol, and 10 µg of protease inhibitor cocktail/ml), phosphorylation reactions were performed at 30°C in 20 mM HEPES-NaOH (pH 7.6), supplemented with 20 µM ATP, 5 µCi of[-³²P]ATP, and 3 µg of glutathione S-transferase (GST)-IB(1-54) substrate (Boston Biologicals, Wellesley, Mass.). Specificity of the kinase reaction was confirmed by using mutant GST-IB(1-54-AA), in which alanines are substituted for serines 32 and 36 (provided by J. DiDonato). The reactions were terminated after 30 min by heat denaturation in the presence of 1% SDS, and radiolabeled phosphoproteins were resolved by SDS-PAGE. The IgG bands on the gels were quantified to control for possible variations among the amounts of immunocomplex loaded in each lane. The gels were then dried at 80°C for 1 h in vacuo. Bands were detected by exposure to Biomax film (Kodak, Rochester, N.Y.) at –80°C.

Preparation of nuclear extracts and EMSA. Nuclear extracts from ECs were prepared, and the presence of DNA-binding activity was analyzed by using an oligonucleotide probe (22 bp, 5'-AGT TGA GGG GAC TTT CCC AGG C-3') containing a consensus NF-B sequence (3, 31). End labeling of the probe was done by using T4 polynucleotide kinase according to the manufacturer's instructions (Promega Corp., Madison, Wis.). Binding reactions for the formation of DNA-protein complexes and subsequent resolution on native polyacrylamide gels (4% [wt/vol]) were performed by using Promega's electrophoretic mobility gel shift assay (EMSA) system (37). Total protein (5 to 8 µg) from extracts prepared for the IP-kinase assay were also used for EMSAs where noted.

Densitometric analysis. The autoradiograms were scanned in the grayscale mode by using a Hewlett-Packard ScanJet scanner at a resolution setting of 300 to 600 dpi. Volume analysis was performed by using ImageQuant software (v. 3.3; Molecular Dynamics, Sunnyvale, Calif.). The results are reported as means ± the standard errors (SE) or standard deviations as noted.

	RESULTS

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Analysis of mRNA and protein expression of the NF-B subunits, p65 and p105/p50. In a previous study, we showed that the major NF-B/DNA complex activated during R. rickettsii infection is the prototypical heterodimer of p65 and p50 DNA-binding subunits (37). The p105 protein is an inactive precursor, which is processed upon cell stimulation to produce the active p50 subunit with DNA-binding capability. Thus, analysis of p50 also represents a measure of the p105 precursor levels (29). The possibility that altered expression of p105/p50 or p65 might play a role in Rickettsia-induced NF-B activation has precedent from studies suggesting that infection of cultured fibroblasts by human cytomegalovirus results in the upregulation of the message for both p105 and p65 through modulation by both viral and cellular factors (48, 49). To determine the pattern of mRNA expression of these subunits, we performed Northern hybridization with specific cDNA probes and total RNA isolated from ECs at 3, 7, 14, and 21 hpi. In three independent experiments, no significant differences in the steady-state level of mRNA for p65 were evident, whereas that of p105/p50 exhibited a slight increase over basal expression (Fig. 1A). Fold differences ranged from (1.23 ± 0.03)- to (1.4 ± 0.2)-fold (mean ± the SE) for p65 and from (1.5 ± 0.3)- to (2.1 ± 0.3)-fold for p105 in R. rickettsii-infected ECs over the 21-h time course compared to uninfected controls. This observation was further confirmed by immunoblot analysis to monitor the protein levels of p65 and p50 in cell lysates obtained from uninfected and infected ECs at various times. In agreement with the mRNA data, no noticeable changes were detected in the steady-state protein levels of p65 (data not shown), and subtle changes in p105/p50 mRNA expression were not reflected by detectable changes in the level of p50 protein (Fig. 1B).

View larger version (49K): [in this window] [in a new window]   FIG. 1. Analysis of steady-state p65 and p50 mRNA and protein levels during R. rickettsii infection. (A) Uninfected ECs (Con) or cells infected with R. rickettsii for the indicated times were processed for the isolation of total RNA. A total of 20µg of RNA for each experimental condition was subjected to Northern blot analysis with p65 and p105/p50 probes. A representative blot for each probe is shown. (B) Equal volumes of total protein lysates prepared from uninfected (Con) or infected (Rr) ECs at different times were resolved by SDS-PAGE electrophoresis and blotted onto nitrocellulose membrane. The blots were probed with a polyclonal anti-p50 antibody, followed by a monoclonal anti--tubulin to control for sample loading.

R. rickettsii infection of ECs activates NF-B via degradation of IB.

View larger version (29K): [in this window] [in a new window]   FIG. 2. IB mRNA expression during R. rickettsii infection. (A) ECs were either left untreated or infected with R. rickettsii for the indicated times prior to isolation of total RNA. A total of 20 µg of RNA for each condition was subjected to Northern blot analysis with an IB probe, followed by analysis with a probe for the housekeeping gene GAPDH. A representative blot is shown. (B) Northern blots (n = 4) were scanned, and band intensities were quantified as described in the text. Values for IB were first normalized to those for GAPDH, and infected conditions were then compared to the corresponding untreated condition at each time. Changes were averaged across all experiments and are presented as the fold change relative to untreated conditions (mean ± the SE).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Western blot analysis of steady-state IB and phospho-IB (Ser-32) protein levels during R. rickettsii infection. (A) Cultures were infected with R. rickettsii (Rr) or left uninfected (Con) for the indicated number of hours prior to preparation of cellular protein extracts. Equal volumes of samples were separated by SDS-PAGE. Blots were sequentially probed with antibodies against phospho-IB, total IB, and -tubulin. A representative blot is shown. Extracts from either untreated or TNF--treated HeLa cells (New England Biolabs) were used as positive controls to identify the appropriate total IB and phospho-IB bands, respectively (not shown). (B) Western blots were scanned and band intensities were quantified as described in the text. Phospho-IB (n = 4) and total IB (n = 3) values were first normalized to the -tubulin values and then compared to values obtained from untreated conditions. Changes were averaged across all experiments and are presented as the fold change relative to untreated conditions (mean ± the SE). Error bars for total IB fall near or within the closed circles used for the datum points.

Activation of IKK and IKKß precedes the Rickettsia-induced NF-B response.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Changes in IKK activity during R. rickettsii infection. (A) Assay for quantitative measurement of IKK activity. ECs were incubated with TNF- (20 ng/ml for 10 min), LPS (E. coli O111:B4; 10 µg/ml for 60 min), or culture medium alone (Con). For each sample, whole-cell extract (20 µg of protein) was subjected to immunoprecipitation with 2 µg each of IKK-specific antibody (lane 1), normal mouse IgG (lane 2, negative control for specificity), or no antibody (lane 3, control for background phosphorylation). The kinase activity of immunoprecipitates was determined by in vitro phosphorylation of GST-IB substrate. The results of a typical IP-kinase assay are shown. (B) R. rickettsii infection activates IKK activity. ECs were infected with rickettsiae for different times corresponding to early and late phases of NF-B activation or stimulated with TNF- (positive control). IKK activity was measured by IP-kinase assay with GST-IB(1-54) substrate. (C) Kinetics of Rickettsia-induced IKK activity. The experiments were performed as described in panel B, and the activity of IKK was analyzed at selected time points during the course of infection. Representative films from independent observations were scanned and quantified densitometrically. In each experiment, the basal activity in uninfected cells was determined by averaging the band intensity units at different times. The induction above the baseline value was then calculated. The results are presented as the mean ± the SE (n 3).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Effect of R. rickettsii infection on IKKß activity. (A) Activation of endogenous IKKß. EC were infected with R. rickettsii organisms for different times corresponding to early and late phases of NF-B activation or stimulated with TNF-. IKKß activity was measured by IP-kinase assay with GST-IB(1-54) substrate. (B) Kinetics of Rickettsia-induced IKKß activity. The activity of IKKß was analyzed by using a separate set of aliquots of cellular lysates described above for Fig. 4.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Inactivation of Rickettsia inhibits infection-induced IKK activity and nuclear translocation of NF-B. Rickettsia organisms were inactivated by heat treatment (65°C for 30 min.) or formaldehyde fixation (3.7% [vol/vol] for 30 min at 25°C). ECs were incubated with viable (Rr), heat-inactivated (Rr Heated), or formalin-fixed (Rr FF) rickettsiae for 2 h, followed by the isolation of total protein extracts. The results for the activities of IKK and IKKß are shown in panels A and B. For comparison, the basal levels of activities in uninfected ECs (Con) were assigned a value of 1. The data presented are the mean ± the SE from three independent experiments. The symbols "" and "#" indicate significant changes (P 0.05) in relation to Con and Rr, respectively. (C) Results of a typical gel retardation assay to investigate activation of NF-B. Nuclear proteins (2.5 µg per condition) were incubated with 32P-labeled consensus NF-B oligonucleotide to measure the extent of DNA-protein binding. The specificity of the binding reactions was ensured by including a 100-fold excess of unlabeled probe (+cold).

View larger version (31K): [in this window] [in a new window]   FIG. 7. Effects of IKK inhibitors on R. rickettsii-mediated IKK and NF-B activation. (A) Parthenolide effectively blocks infection-induced IKK activity. After preincubation with medium alone (Con) or with medium containing aspirin (+ASP) and parthenolide (+PAR) for 30 min, ECs were left uninfected, exposed to TNF-, or infected with R. rickettsii for 2 h. IKK activity is presented as the fold induction relative to the basal level in untreated, uninfected cells (Con). The values are the mean ± the SE from three independent experiments with the exception of TNF+ASP (n = 2). The P values for different experimental conditions relative to unstimulated, infected, and TNF--induced cells are also shown. (B) Aspirin inhibits Rickettsia-induced IKKß kinase activity. The activity of IKKß was measured after infection in the presence or absence of aspirin and parthenolide. (C) Effect of specific IKK inhibitor parthenolide (PAR) on R. rickettsii-induced NF-B activation. Nuclear protein extracts from uninfected control (Con) and ECs infected in the absence (Rr) or presence of PAR (Rr+PAR) for 3 h were subjected to EMSA. The positions of gel-shifted complex for NF-B, nonspecific complex (NS), and excess free probe are indicated. The specificity of the complex formation was confirmed by incubation with a 100-fold molar excess of unlabeled probe during DNA-protein binding (+cold). Similar results were obtained with aspirin treatment.

Inhibitors of IKK block R. rickettsii-induced NF-B activation. To further define the role of IKK and IKKß in upstream signaling, the effects of two structurally different anti-inflammatory agents on R. rickettsii-mediated NF-B activation were investigated. Parthenolide, a sesquiterpene lactone compound, predominantly targets the IKK complex (16), and aspirin has been shown to inhibit IKKß activity (27). To confirm specificity in our culture system and to determine an effective concentration for subsequent infection experiments, ECs were stimulated with TNF- in the presence or absence of increasing concentrations of parthenolide (5 to 20 µM), and whole-cell extracts were analyzed in parallel for NF-B DNA binding (not shown) and IKK activation (Fig. 7A). Both of these responses were inhibited by parthenolide in a dose-dependent manner, with complete blockade at concentrations of 10 µM or higher. The optimum concentration of aspirin to achieve complete inhibition of TNF--induced NF-B and IKK activation was 10 mM. Further experiments with R. rickettsii were carried out with aspirin at a final concentration of 10 mM and parthenolide at 10 µM, since these treatments had no significant effect on the extent of infection as assessed by indirect immunofluorescence microscopy. Although incubation with these compounds alone caused a slight induction of IKK activity in ECs, the apparent changes were found to be statistically insignificant (Fig. 7A and B) and did not result in an increased nuclear translocation of NF-B (Fig. 7C). Parthenolide treatment had an inhibitory effect on both R. rickettsii-induced IKK, as well as IKKß activities, as measured at 2 hpi, although its effect on IKK was more pronounced. Aspirin was also able to inhibit infection-induced activation of IKK and IKKß. Furthermore, inhibitory effects of aspirin and parthenolide on infection-induced IKK activities were paralleled by a significant reduction (>75%) or complete attenuation of NF-B DNA-binding activity due to R. rickettsii infection (Fig. 7). Together, these results clearly indicate that NF-B activation due to infection occurs predominantly through upstream signaling mechanisms leading to the activation of IKK and IKKß.

	DISCUSSION

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Activation of NF-B plays a critical role in the pathophysiology of R. rickettsii infection by ensuring the survival of infected host cells to allow for the continued growth and replication of intracellular bacteria (3, 22). A particularly intriguing aspect of Rickettsia-induced NF-B activation is that it occurs in two distinct phases during infection of cultured ECs (37); there is also a possibility that differences may exist in the upstream signaling mechanisms contributing to this biphasic pattern. The goal of the present study was to define changes in the mRNA and protein expression of major NF-B subunits, degradation of IB proteins, and activation of catalytic components of the IB kinase complex, a cytoplasmic signalosome responsible for the phosphorylation of IB and/or IBß.

Although NF-B is known to participate in its own regulation by partially regulating the expression of the human p105/p50 gene promoter (48), the steady-state mRNA of p105 and protein levels of p50 in Rickettsia-infected cells remained relatively unaltered. This suggests that de novo transcription via autoregulatory or other transcription factors (e.g., AP1 and SP1) does not take place during the course of infection. It also indicates that infection did not affect the processing of the p105 precursor to the active p50 form. Similar to other cell types, p65 was expressed constitutively, and R. rickettsii infection did not appear to alter its expression profile. That the p65 promoter contains at least three functional sites for the transcription factor SP1 upstream of the initiation site, but none for NF-B (48, 49), provides further indirect evidence for noninvolvement of SP1-mediated transcriptional events and supports the notion that SP1 is not activated during R. rickettsii infection (L. A. Sporn and S. K. Sahni, unpublished observations).

The IB family proteins can differentially associate with and regulate the activity of various NF-B dimers in the cytoplasm. The targeted degradation of these proteins is critical to all known mechanisms of NF-B activation. In R. rickettsii-infected ECs, the early peak of NF-B DNA-binding activity coincided with increased IB mRNA levels and was preceded by increased phosphorylation of IB (Fig. 2 and 3). Lower levels of total protein further indicate concurrent degradation of phosphorylated IB (Fig. 3). This is in agreement with our earlier findings that inhibition of proteasomal IB degradation via MG132 or MG115 or expression of a dominant-negative IB can effectively inhibit the early phase of Rickettsia-induced NF-B in ECs or fibroblasts, respectively (3, 31). The failure to accumulate IB back to basal levels at all times despite evidence for increased mRNA expression suggests the involvement of continued IB degradation, at least in part, in the late phase of NF-B activation. It is possible that other unknown mechanisms governing posttranscriptional modifications to either inhibit or downregulate the resynthesis of IB, which are known to occur during cytomegalovirus infection of monocytes (50) and respiratory syncytial virus infection of A549 cells (21), may also be involved.

In spite of significant sequence homology and ability to interact with the same set of NF-B proteins, IB and IBß display distinct responses to different inducers. Persistent NF-B activation by soluble agonists (e.g., LPS and IL-1), infection with pathogenic bacteria (Listeria), and pathogen-derived mediators (Tax protein of human T-cell leukemia virus type 1) usually involve prolonged activation of NF-B by binding to hypophosphorylated IBß or inactivation of IBß (15, 28, 39). We observed only subtle changes in the mRNA and protein expression levels of IBß at all times tested in the present study, suggesting that it does not contribute appreciably to NF-B activation during R. rickettsii infection. IB, another member of the IB family, associates predominantly with p65 homodimers or p65-cRel heterodimers in the cytoplasm but not with p50 containing heterodimers. It has been proposed that specific interactions of IB with c-Rel dimers play a functional role in the induction of expression of adhesion molecules in vascular ECs (36), and degradation of both IB and IB to various degrees occurs during infection of different intestinal ECs with enteroinvasive bacteria (7). Although R. rickettsii infection of ECs triggers nuclear translocation of p65 without affecting c-Rel (37), a possibility which remains to be investigated is that the degradation of IB associated with p65 (RelA) homodimers may contribute to the second phase of NF-B activation during rickettsial infection.

Recent studies have established the importance of IKK complex phosphorylation in the canonical pathway of NF-B activation in response to a variety of factors, including cytokines, pathogenic bacteria, and viruses (2, 33, 46). Increased kinase activity of both IKK and IKKß as a consequence of R. rickettsii infection clearly demonstrates modulation of the IKK complex in ECs. Early during the infection, the levels of activation for both kinases were strikingly similar and coincided with the Ser32 phosphorylation of IB, an essential signaling step preceding nuclear translocation of NF-B. Thus, it appears that the early transient phase of Rickettsia-induced NF-B activation is mediated by mechanisms involving both IKK and IKKß. IKK activation at later times apparently corresponded with the second phase of NF-B, but only IKK activity achieved levels comparable to those seen early during the infection. The role of IKK in the late sustained phase of NF-B activation, however, is unique in that existing biochemical and genetic evidence indicates that, whereas IKKß serves as the dominant kinase, IKK performs a potentially redundant function in stimulus-induced NF-B activation (4). It is also possible that NIK (for NF-B inducing kinase) or some other upstream mitogen-activated protein kinase(s) may be activated, similar to those stimulated by cytokines. Chlamydia pneumoniae infection of monocytic cells also involves increased activity of IKK and IB/IB proteolysis (6), but the activation of IKK in this case is transient and does not resemble the pattern seen in our studies. It should be noted that intracellular R. rickettsii organisms reside and replicate mainly in the host cell cytosol (8, 42), and studies using a "cell-free" system have documented the ability of viable R. rickettsii to directly interact with and enhance the DNA-binding activity of NF-B, which otherwise remains inactive in cytoplasmic extracts isolated from ECs (31). Such activation of NF-B occurs in a proteasome-independent fashion and requires a rickettsial protease activity (31, 32). Thus, it is possible that the second phase of NF-B activation during infection of intact cells may occur due to the combination of cellular activation of IKK and enhanced direct interactions between either intracellular rickettsiae or a rickettsial factor with inactive cytoplasmic NF-B.

The specific interactions of Rickettsia organisms with the host EC surface to orchestrate the events resulting in IKK/NF-B activation remain to be elucidated in further detail. Thus far, the identities of a potential host cell receptor or a major rickettsial component(s) serving to trigger the upstream signaling pathways are not known. Initial examination with inactivated R. rickettsii revealed that the exposure of host cell surface to live bacteria is necessary and a heat-sensitive protein is responsible for the onset of endothelial activation. In R. rickettsii, the major immunodominant surface-exposed proteins are the outer membrane proteins OmpA and OmpB, of which OmpA is critical for initial adhesion to the host cell membrane (26). Further investigation of OmpA involvement in infection-induced cell signaling is particularly important in light of the finding that outer membrane proteins of Bartonella henselae are major pathogenic factors for the production of EC responses (11). Bacterial LPS is known to be a strong inducer of NF-B in a variety of cell types (2, 12). Although it remains a possibility, a potential role for rickettsial LPS is somewhat unlikely considering its significantly lower endotoxin activity in comparison to classically active LPS from Escherichia coli or Salmonella spp. (13) and the existing evidence for noninvolvement in in vitro endothelial responses characterized thus far (30, 31, 34). Moreover, recent data from our laboratory show that adsorption of LPS by incubation with either polymyxin B-agarose beads (30) or a monoclonal antibody to R. rickettsii LPS has no effect on the activation of IKK. A crude preparation of rickettsial LPS, which was much less effective in inducing the expression and activity of tissue factor in cultured ECs as expected, also had no apparent effect on IKK activity, whereas E. coli (O111:B4) LPS triggered potent induction of both tissue factor and IKK activities (Fig. 4) (S. K. Sahni and E. Rydkina, unpublished observations). Another possibility being explored in our laboratory is that R. rickettsii-induced endothelial signaling, especially late during infection, may be an indirect response due to the production of secondary inducers such as cytokines, e.g., TNF- or IL-1.

Since NF-B plays a central role in acute and chronic inflammation, signaling pathways that regulate its activity have become a focal point as molecular targets for the development of new therapeutic compounds (23). Activation of NF-B is essential for preventing apoptosis and ensuring the survival of ECs during R. rickettsii infection and plays an important role in triggering infection-induced responses of the vascular endothelium by stimulating the expression of chemokines IL-8 and monocyte chemoattractant protein 1 (3; D. R. Clifton, H. Huyck, G. Pryhuber, R. S. Freeman, L. A. Sporn, and S. K. Sahni, Abstr. Am. Soc. Rickettsiol., abstr. 79, p. 58, 2001). Thus, inhibition of IKK and NF-B by using nonsteroidal anti-inflammatory agents may provide attractive targets for novel antirickettsial therapies. Our data suggest that inhibition of R. rickettsii-induced activation of IKK and IKKß with aspirin or parthenolide is sufficient to effectively attenuate the early transient NF-B response. This may reduce the ensuing inflammatory reactions and risk of vascular dysfunction associated with rickettsial infections. Sesquiterpene lactones such as parthenolide are amenable for use in treating infections and inflammation of the skin and other organs (18). Studies have also shown that aspirin exhibits previously unrecognized antibacterial effects mediated by reduction of hematogenous bacterial dissemination and embolism during Staphylococcus aureus endocarditis (24) and inhibition of chlamydial growth in human ECs (47).

In summary, infection of human ECs with viable R. rickettsii induces IKK and IKKß activation, an important signaling event which leads to the phosphorylation and proteolysis of IB and translocation of active NF-B dimers to the nucleus. The steady-state expression levels of key NF-B proteins p65 and p50 and inhibitor protein IBß, however, remain relatively unchanged. The anti-inflammatory compounds aspirin and parthenolide inhibit Rickettsia-induced IKK phosphorylation of IB and nuclear translocation of NF-B, suggesting that at least the early signaling occurs through this established pathway. Such action could effectively disrupt the production of NF-B-dependent inflammatory mediators. These findings thus provide new insights into the molecular basis of NF-B regulation during R. rickettsii infection and suggest that newly developed specific inhibitors of IKK activity may not only serve as anti-inflammatory agents but also as efficacious therapies to prevent vascular damage and reduce morbidity in human infections.

	ACKNOWLEDGMENTS

 
We thank Li Hua Rong for cultures of ECs; David J. Silverman for providing R. rickettsii; Edward Schwarz for providing plasmids containing IB, IBß, and p65 cDNA; Albert Baldwin, Jr., for providing the plasmid containing p105/p50 cDNA; P. J. Simpson-Haidaris for providing the GAPDH probe for Northern analysis; David H. Walker for providing monoclonal antibody to R. rickettsii LPS; and Minh-Doan T. Nguyen and Brian J. Rybarczyk for helpful discussions.

This study was supported in part by grants AI40689 and HL30616 from the National Institutes of Health, Bethesda, Md.

	FOOTNOTES

 
* Corresponding author. Mailing address: Hemostasis and Thrombosis Program, Hematology-Oncology Unit, Department of Medicine, P.O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Phone: (585) 275-0439. Fax: (585) 473-4314. E-mail: Sanjeev_sahni{at}urmc.rochester.edu.

Editor: J. B. Bliska

Present address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1.	Baldwin, A. S., Jr. 2001. The transcription factor NF-B and human disease. J. Clin. Investig. 107:3-6.[CrossRef][Medline]
2.	Bierhaus, A., and P. P. Naworth. 2003. Modulation of the vascular endothelium during infection: the role of NF-B activation. Contrib. Microbiol. 10:86-105.[Medline]
3.	Clifton, D. R., R. A. Goss, S. K. Sahni, D. van Antwerp, R. B. Baggs, V. J. Marder, D. J. Silverman, and L. A. Sporn. 1998. NF-B-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection. Proc. Natl. Acad. Sci. USA 95:4646-4651.[Abstract/Free Full Text]
4.	Denk, A., M. Goebeler, S. Schmid, I. Berberich, O. Ritz, D. Lindemann, S. Ludwig, and T. Wirth. 2001. Activation of NF-B via the IB kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells. J. Biol. Chem. 276:28451-28458.[Abstract/Free Full Text]
5.	DiDonato, J. A. 2000. Assaying for IB kinase activity. Methods Enzymol. 322:393-400.[Medline]
6.	Donath, B., C. Fischer, S. Page, S. Prebeck, N. Jilg, M. Weber, C. da Costa, D. Neumeier, T. Miethke, and K. Brand. 2002. Chlamydia pneumoniae activates IKK/IB-mediated signaling, which is inhibited by 4-HNE and following primary exposure. Atherosclerosis 165:79-88.[CrossRef][Medline]
7.	Elewaut, D., J. A. DiDonato, J. M. Kim, F. Truong, L. Eckmann, and M. F. Kagnoff. 1999. NF-B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J. Immunol. 163:1457-1466.[Abstract/Free Full Text]
8.	Eremeeva, M. E., G. A. Dasch, and D. J. Silverman. 2000. Interaction of rickettsiae with eukaryotic cells: adhesion, entry, intracellular growth, and host cell responses. Subcell. Biochem. 33:479-516.[Medline]
9.	Fan, C., Q. Li, Y. Zhang, X. Liu, M. Luo, D. Abbott, W. Zhou, and J. F. Engelhardt. 2004. IB and IBß possess injury context-specific functions that uniquely influence hepatic NF-B induction and inflammation. J. Clin. Investig. 113:746-755.[CrossRef][Medline]
10.	Fischer, C., S. Page, M. Weber, T. Eisele, D. Neumeier, and K. Brand. 1999. Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IB kinase signalosome activation and IB proteolysis. J. Biol. Chem. 274:24625-24632.[Abstract/Free Full Text]
11.	Fuhrmann, O., M. Arvand, A. Gohler, M. Schmid, M. Krull, S. Hippenstiel, J. Seybold, C. Dehio, and N. Suttorp. 2001. Bartonella henselae induces NF-B-dependent upregulation of adhesion molecules in cultured human endothelial cells: possible role of outer membrane proteins as pathogenic factors. Infect. Immun. 69:5088-5097.[Abstract/Free Full Text]
12.	Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225-260.[CrossRef][Medline]
13.	Hackstadt, T. 1996. The biology of rickettsiae. Infect. Agents Dis. 5:127-143.[Medline]
14.	Haidaris, P. J., T. W. Wright, F. Gigliotti, and C. G. Haidaris. 1992. Expression and characterization of a cDNA clone encoding an immunodominant surface glycoprotein of Pneumocystis carinii. J. Infect. Dis. 166:1113-1123.[Medline]
15.	Hauf, N., W. Goebel, F. Fiedler, Z. Sokolovic, and M. Kuhn. 1997. Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-B (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IB and IBß degradation. Proc. Natl. Acad. Sci. USA 94:9394-9399.[Abstract/Free Full Text]
16.	Hehner, S. P., T. G. Hofmann, W. Dröge, and M. L. Schmitz. 1999. The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-B by targeting the IB kinase complex. J. Immunol. 163:5617-5623.[Abstract/Free Full Text]
17.	Heilker, R., F. Freuler, M. Vanek, R. Pulfer, T. Kobel, J. Peter, H.-G. Zerwes, H. Hofstetter, and J. Eder. 1999. The kinetics of association and phosphorylation of IB isoforms by IB kinase 2 correlate with their cellular regulation in human endothelial cells. Biochemistry 38:6231-6238.[CrossRef][Medline]
18.	Heinrich, M., M. Robles, J. E. West, B. R. Ortiz de Montellano, and E. Rodriguez. 1998. Ethnopharmacology of Mexican asteraceae (Compositae). Annu. Rev. Pharmacol. Toxicol. 38:539-565.[CrossRef][Medline]
19.	Heinzen, R. A. 2003. Rickettsial actin-based motility: behavior and involvement of cytoskeletal regulators. Ann. N. Y. Acad. Sci. 990:535-547.[Abstract/Free Full Text]
20.	Hirano, F., M. Chung, H. Tanaka, N. Maruyama, I. Makino, D. D. Moore, and C. Schedereit. 1998. Alternative splicing variants of IBß establish differential NF-B signal responsiveness in human cells. Mol. Cell. Biol. 18:2596-2607.[Abstract/Free Full Text]
21.	Jamaluddin, M., A. Casola, R. P. Garofalo, Y. Han, T. Elliott, P. L. Ogra, and A. R. Brasier. 1998. The major component of IB proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells. J. Virol. 72:4849-4857.[Abstract/Free Full Text]
22.	Joshi, S. G., C. W. Francis, D. J. Silverman, and S. K. Sahni. 2003. Nuclear factor-B protects against host cell apoptosis during Rickettsia rickettsii infection by inhibiting activation of apical and effector caspases and maintaining mitochondrial integrity. Infect. Immun. 71:4127-4136.[Abstract/Free Full Text]
23.	Karin, M., Y. Yamamoto, and Q. M. Wang. 2004. The IKK NF-B system: a treasure trove for drug development. Nat. Rev. Drug Discov. 3:17-26.[CrossRef][Medline]
24.	Kupferwasser, L. I., M. R. Yeaman, S. M. Shapiro, C. C. Nast, P. M. Sullam, S. G. Filler, and A. S. Bayer. 1999. Acetylsalicylic acid reduces vegetation bacterial density, hematogenous bacterial dissemination, and frequency of embolic events in experimental Staphylococcus aureus endocarditis through antiplatelet and antibacterial effects. Circulation. 99:2791-2797.[Abstract/Free Full Text]
25.	Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
26.	Li, H., and D. H. Walker. 1998. rOmpA is a critical protein for the adhesion of Rickettsia rickettsii to host cells. Microb. Pathog. 24:289-298.[CrossRef][Medline]
27.	Maeda, S., H. Yoshida, K. Ogura, Y. Mitsuno, Y. Hirata, Y. Yamaji, M. Akanuma, Y. Shiratori, and M. Omata. 2000. Helicobacter pylori activates NF-B through a signaling pathway involving IB kinases, NF-B-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells. Gastroenterology 119:97-108.[CrossRef][Medline]
28.	McKinsey, T. A., J. A. Brockman, D. C. Scherer, S. W. Al-Murrani, P. L. Green, and D. W. Ballard. 1996. Inactivation of IBß by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-B. Mol. Cell. Biol. 16:2083-2090.[Abstract]
29.	Orian, A., A. L. Schwartz, A. Israel, S. Whiteside, C. Kahana, and A. Ciechanover. 1999. Structural motifs involved in ubiquitin-mediated processing of the NF-B precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain. Mol. Cell. Biol. 19:3664-3673.[Abstract/Free Full Text]
30.	Rydkina, E., A. Sahni, D. J. Silverman, and S. K. Sahni. 2002. Rickettsia rickettsii infection of cultured human endothelial cells induces heme oxygenase 1 expression. Infect. Immun. 70:4045-4052.[Abstract/Free Full Text]
31.	Sahni, S. K., D. Van Antwerp, D. J. Silverman, V. J. Marder, and L. A. Sporn. 1998. Proteasome-independent activation of nuclear factor-B in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii. Infect. Immun. 66:1827-1833.[Abstract/Free Full Text]
32.	Sahni, S. K., E. Rydkina, S. G. Joshi, L. A. Sporn, and D. J. Silverman. 2003. Interactions of Rickettsia rickettsii with endothelial nuclear factor-B (NF-B) in a "cell-free" system. Ann. N. Y. Acad. Sci. 990:635-641.[Free Full Text]
33.	Santoro, M. G., A. Rossi, and C. Amici. 2003. NF-B and virus infection: who controls whom. EMBO J. 22:2552-2560.[CrossRef][Medline]
34.	Shi, R.-J., P. J. Simpson-Haidaris, N. B. Lerner, V. J. Marder, D. J. Silverman, and L. A. Sporn. 1998. Transcriptional regulation of endothelial cell tissue factor expression during Rickettsia rickettsii infection: involvement of the transcription factor NF-B. Infect. Immun. 66:1070-1075.[Abstract/Free Full Text]
35.	Simeonidis, S., D. Stauber, G. Chen, W. A. Hendrickson, and D. Thanos. 1999. Mechanisms by which IB proteins control NF-B activity. Proc. Natl. Acad. Sci. USA 96:49-54.[Abstract/Free Full Text]
36.	Spiecker, M., H. Darius, and J. K. Liao. 2000. A functional role of IB in endothelial cell activation. J. Immunol. 164:3316-3322.[Abstract/Free Full Text]
37.	Sporn, L. A., S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab. 1997. Rickettsia rickettsii infection of cultured human endothelial cells induces NF-B activation. Infect. Immun. 65:2786-2791.[Abstract]
38.	Suyang, H., R. Phillips, I. Douglas, and S. Ghosh. 1996. Role of unphosphorylated, newly synthesized IBß in persistent activation of NF-B. Mol. Cell. Biol. 16:5444-5449.[Abstract]
39.	Thompson, J. E., R. J. Phillips, H. Erdjument-Bromage, P. Tempst, and S. Ghosh. 1995. IBß