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Am J Physiol Lung Cell Mol Physiol 289: L536-L544, 2005. First published June 10, 2005; doi:10.1152/ajplung.00164.2005
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Prevention of NF-{kappa}B activation in vivo by a cell-permeable NF-{kappa}B inhibitor peptide

Ana L. Mora,1,2,3 John LaVoy,1,2 Martha McKean,1,2 Arlene Stecenko,1,2,3 Kenneth L. Brigham,1,2,3 Richard Parker,1,2 and Mauricio Rojas1,2,4

1Division of Pulmonary, Allergy, and Critical Care Medicine, 2Center for Translational Research in the Lung, and 3McKelvey Center for Lung Transplantation, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and 4Department of Medicine, Vanderbilt University, Nashville, Tennessee

Submitted 12 April 2005 ; accepted in final form 6 June 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The NF-{kappa}B/Rel transcription factor family plays a central role in coordinating the expression of a variety of genes that regulate stress responses, immune cell activation, apoptosis, proliferation, differentiation, and oncogenic transformation. Interventions that target the NF-{kappa}B pathway may be therapeutic for a variety of pathologies, especially immune/inflammatory diseases. Using membrane translocating sequence (MTS) technology, we developed a cell-permeable dominant inhibitor of NF-{kappa}B activation, termed I{kappa}B{alpha}-({Delta}N)-MTS. This molecule contains a 12-amino acid MTS motif attached to the COOH-terminal region of a nondegradable inhibitor protein [I{kappa}B{alpha}-({Delta}N)]. The recombinant protein enters cells and localizes in the cytoplasm. Delivery of the I{kappa}B{alpha}-({Delta}N)-MTS to cell lines and primary cells inhibited nuclear translocation of NF-{kappa}B proteins induced by cell activation. The protein also effectively inhibited NF-{kappa}B activation in vivo in two different animal models: NF-{kappa}B activation in response to skin wounding in mice and NF-{kappa}B activation in lungs after endotoxin treatment in sheep. Inhibition of NF-{kappa}B by the I{kappa}B{alpha}-({Delta}N)-MTS in the endotoxin model attenuated physiological responses to endotoxemia. These data demonstrate that activation of NF-{kappa}B can be inhibited using a recombinant protein designed to penetrate into cells. This technology may provide a new approach to NF-{kappa}B pathway-targeted therapies.

drug delivery; therapy; cell-permeable protein; inflammation; lung injury

THE INDUCIBLE TRANSCRIPTION FACTOR NF-{kappa}B plays a central role in regulation of immune, inflammatory, and carcinogenic responses. Whereas normal activation of NF-{kappa}B is required for cell survival and immunity, nonregulated NF-{kappa}B activation may cause several pathological states, especially those characterized by persistent acute inflammation. Responses to a wide variety of stimuli are mediated through NF-{kappa}B signaling pathways.

This family of transcription factors includes p50, p52, c-Rel, RelB, and p65 (Rel A). In a resting state, NF-{kappa}B proteins are sequestered in the cytoplasm as homo- or heterodimer (usually the heterodimer p50/p65) by association with I{kappa}B inhibitory proteins (I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon}) (1, 46). I{kappa}B{alpha}, the best-characterized I{kappa}B molecule, contains serines at positions 32 and 36 that are phosphorylated by the activation complex of I{kappa}B kinases (IKK), separated from NF-{kappa}B, and then ubiquitinated, targeted to proteasomes, and degraded. Removal of I{kappa}B{alpha} from the complex exposes a nuclear localization sequence on NF-{kappa}B dimers that translocates into the nucleus (8, 15, 20) and binds to specific DNA sequences modulating expression of >150 target genes. The large majority of proteins encoded by these genes participates in immune responses. These include cytokines, receptors, adhesion molecules, and adaptor proteins (2).

Numerous efforts have been made to develop regulators of NF-{kappa}B activity (13, 33, 51). In theory, NF-{kappa}B activation could be inhibited in several ways: 1) blocking incoming signal pathways that activate the IKK complex; 2) interfering in the phosphorylation, ubiquitination, and degradation of I{kappa}B proteins; or 3) blocking the translocation of NF-{kappa}B dimers into the nucleus by targeting the nuclear pore protein complex. Only strategies that target the I{kappa}B molecules are likely to be specific for the NF-{kappa}B pathway.

To affect intracellular signaling, inhibitors must access the cell interior, and one strategy for achieving that has been to employ a membrane translocating sequence (MTS). MTS is a 12-amino acid residue sequence that, when fused to peptides or proteins, transports them through the cell membrane (11, 21–23, 35–37). MTS was originally derived from the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor (K-FGF). We modified the K-FGF export signal to create an MTS that functions as a cellular import signal. Fusion of this sequence to either the NH2 or the COOH terminus of a protein renders the fusion protein cell membrane permeable. Our previous work shows that the MTS can efficiently deliver up to several million molecules per cell of a large variety of proteins (14). Proteins have been delivered successfully into many cell types with preservation of their function. Immunofluorescence and confocal imaging studies indicate the imported proteins are evenly distributed and stable in the cell and are not cytotoxic (11, 19).

To determine whether this technology could be used to modify NF-{kappa}B-mediated cellular responses, we created a fusion protein containing a modified I{kappa}B{alpha} protein [termed I{kappa}B{alpha}-({Delta}N)] that cannot be phosphorylated and therefore functions as a dominant negative NF-{kappa}B inhibitor. We found that the permeable I{kappa}B{alpha}-({Delta}N)-MTS fusion protein inhibited nuclear translocation of NF-{kappa}B complex in cell culture and in vivo and attenuated NF-{kappa}B-dependent physiological responses in vivo.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Sheep were housed and cared for in the Emory University Division of Animal Resources in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised 1996). Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of Emory University.

Mice were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised 1996). Procedures in mice were approved by the Institutional Animal Care and Use Committee of Vanderbilt University.

Design of I{kappa}B{alpha}-({Delta}N)-MTS fusion protein. cDNA from Jurkat T cells was used as a template to amplify specific I{kappa}B{alpha} cDNA (16). Each of the primers contained a BamHI site at the 5' end. The sequence of the primers was: 5'-CCGGATCCCCATGAAAGACGAGGAGTACGAGCAGATGGTC-3' and 5'-CCGGATCCCTAACGTCAGACGCTGGCCTCCAAACACACA-3'. These primers encode a cDNA for an NH2-terminal truncated form (amino acid 37–317) of I{kappa}B{alpha}. The PCR product was originally cloned in pGEMT-easy (Promega) and then subcloned into the MTS expression vector to produce a cell-permeable fusion protein containing a GST (glutathione-S-transferase) tag; we called this molecule I{kappa}B{alpha}-({Delta}N)-MTS (35). As a control, we also produced a permeable GST-MTS and the nonpermeable GST and GST-I{kappa}B{alpha}-({Delta}N) without the MTS sequence in the expression vector pGEX-3X. Translation and reading frame were confirmed by automated sequencing.

Purification of recombinant proteins. The MTS-containing GST fusion proteins were purified using an AKTA-fast-performance liquid chromatography (FPLC). Twenty milligrams of 90% pure recombinant permeable proteins were obtained from 4 l of bacterial culture in a single chromatography run. To reduce endotoxicity, proteins for this study were expressed in Escherichia coli BL21 (DE3) cells inactive for the LPS gene (10). Recombinant cells were grown to optical density 0.7 and induced by the addition of 0.5 mM isopropylthiogalactoside (Sigma). Cells were allowed to grow at 25°C for 4 h and were harvested by centrifugation. The cells were incubated for 30 min at 4°C in lysis buffer [PBS containing 10 mM DTT, 1 mM EDTA, 1 mM PMSF, 0.02% lysozyme (Sigma)]. DNase I (10 µg/ml, Sigma) was added to the lysate and allowed to incubate at room temperature for 30 min. Triton X-100 was added to 1% final concentration, and the solution was allowed to incubate at room temperature for an additional 30 min. The lysate was diluted 1:1 with lysis buffer and applied to a 5-ml GST column (Amersham Biosciences). With the use of an AKTA purifier FPLC system (Amersham Biosciences), the column was washed with PBS and eluted by step gradient with PBS containing 20 mM glutathione. Fractions containing protein were pooled, diluted 1:5 with PBS, applied to a 1-ml HiTrap Q column (Amersham Biosciences), washed with PBS (buffer A), and eluted by linear gradient with PBS containing 2 M NaCl (buffer B). Fractions were analyzed by SDS-PAGE and stored in 10% glycerol at –80°C for later use.

Proteins requiring immediate use were dialyzed in PBS or passed through a desalting column. Endotoxin was removed from protein solutions using a Triton X-114 extraction procedure (24). The procedure was repeated until endotoxin was reduced to acceptable levels. After Triton X-114 extraction, protein concentration and levels of endotoxin (limulus assay) were determined. Fractions were combined and kept at 4°C for short-term storage or at –80°C for long-term storage. The yield of the permeable protein was 20–30% lower than the nonpermeable protein.

Indirect immunofluorescence assay. NIH/3T3 cells were cultured in four-well slides (Nunc) for 3 days at 37°C. Subconfluent cells were washed with media without sera and incubated with the different proteins at a concentration of 100 µg/ml for 1 h at 37°C. The cells were washed with PBS and fixed with 3.7% paraformaldehyde in PBS at 37°C for 15 min. Cells were washed again three times with PBS and treated with 0.25% Triton X-100 in PBS for 10 min. Then, cells were incubated with blocking solution [PBS + 1% normal goat serum (Sigma)] plus 1% BSA (Sigma) for 30 min at 37°C and 5% CO2. After being blocked, cells were incubated with anti-GST (Santa Cruz Biotechnology) in PBS plus 1% BSA for 2 h. Cells were washed three times with PBS and blocked for 30 min with blocking solution. Intracellular protein-antibody complexes were detected by being incubated for 1 h with goat anti-rabbit IgG labeled with Texas red (Molecular Probes). Coverslips were mounted in ProLong antifade (Molecular Probes) and analyzed in a confocal microscope (Zeiss).

Western blot. After delivering the I{kappa}B{alpha}-({Delta}N)-MTS protein, Jurkat T cells were activated with a combination of 50 ng/ml of PMA and 1 µM ionomycin. Activated cells were harvested, and cytoplasmic extracts were prepared. Protein concentration was determined with a colorimetric assay (Bio-Rad). Equal amounts of proteins were mixed with a prewarmed sample buffer. Proteins were separated by SDS-page, transferred to a polyvinylidene difluoride membrane (Amersham), and probed with anti-p50 and -p65 antibody (Santa Cruz Biotechnology). Antigen-antibody complexes were detected with an anti-rabbit serum coupled to horseradish peroxidase (Santa Cruz Biotechnology) and developed by using an enhanced chemiluminescence system (Pierce).

EMSA. A total of 1 x 106 primary thymocytes from C57BL/6 mice were incubated with 50 or 100 µg/ml of the protein in a total volume of 1 ml and activated with PMA/ionomycin, and lung tissue samples obtained from a live sheep treated intravenously with I{kappa}B{alpha}-({Delta}N)-MTS 1 h before endotoxin (see Open-chest sheep preparation) were used to prepare cell extracts. Gel mobility shift assays of NF-{kappa}B/Rel proteins were performed as previously described using a double-stranded 32P-labeled oligonucleotide modified from {kappa}B enhancer sequences in the IL-2 receptor-{alpha} promoter (upper strand, 5'-CAACGGCAGGGGAATTCCCCTCTCCTT) (3, 6, 26, 27, 28). DNA binding reaction mixtures (20 µl) containing 4 µg of nuclear extract, 2 µg of double-stranded poly(dI-dC), 10 µg of BSA buffered in 20 mM HEPES (pH 7.9), 5% glycerol, 1 mM EDTA, 1% Nonidet P-40, and 5 mM DTT were then resolved on native 5% polyacrylamide gels and visualized by autoradiography.

DNA incorporation assay. DO11 T cell receptor (TCR) transgenic mice were used as a source of T cells specific for a known antigen, an ovalbumin peptide (OVA323–339) (31). Antigen-presenting cells (APC) were obtained from adherent spleen cells isolated from wild-type BALB/c mice. DO11 CD4+ T cells were obtained by negative selection using anti-CD8+ and anti-class II magnetic coated beads (Miltenyi). Purified CD4+ cells were incubated at 37°C with 250 µg/ml of the permeable recombinant protein. After 1 h of treatment, cells were washed twice with sterile PBS, and 1 x 105 cells/well were plated simultaneously with 2 x 105 APC cells. OVA323–339 peptide was used at concentrations of 0.1, 0.5, and 1 µg/ml, and cultures were maintained for 48 h at 37°C in 5% CO2. [3H]thymidine (0.1 µg/well) was added, and cells were cultured for an additional 12 h. Thymidine incorporation was measured in a beta counter, and data are presented as counts per minute.

In vivo measurement of luciferase gene expression by bioluminescence. HLL [human immunodeficiency virus (HIV)-long terminal repeat (LTR)/luciferase] transgenic mice were kindly provided by Dr. F. Yull (Vanderbilt Univ.). These reporter transgenic mice have luciferase expression driven by NF-{kappa}B-dependent portion of the HIV-1 LTR (4, 39, 40). Mice were anesthetized, and the abdomen was shaved. Two 1.5-cm-long superficial incisions were made in the skin of the upper and lower portions of the abdomen. The incisions were closed with two stitches using a 4–0 suture. Fusion proteins were administrated intraperitoneally 2 and 12 h after surgery (250 µg/dose in 0.5 ml of PBS). Imaging was obtained using luciferin (50 µg/mouse in 100 µl of PBS) administered intravenously, and mice were imaged with an intensified charge-coupled device (ICCD) camera (model C2400-32; Hamamatsu). This system consists of an image intensifier coupled to an eight-bit CCD camera, allowing for 256 intensity levels for each pixel. For the duration of photon counting, mice were placed inside a light tight box that also houses the camera. Light emission from the mouse was detected using the ICCD camera and customized image processing hardware and software (Hamamatsu). Quantitative analysis was accomplished by defining a standard area (region of interest) in the eight-bit intensity image corresponding to the region of the abdomen overlying the incision and determination of total integrated photon intensity over the region of interest. For presentation, a four-bit (16 intensity levels) digital false-color photon emission image was generated for each captured image according to the same false-color scale. To visualize the dimmer parts of the image, the brighter pixels in the images are displayed as white (thus appearing saturated); however, detected light emission for each image was well below the saturation limit for the camera.

Open-chest sheep preparation. Yearling sheep were anesthetized with thiopental, intubated, secured in a dorsal decubitus position, and ventilated with 3% isoflurane and equal concentrations of compressed air and oxygen. A catheter was inserted into the carotid artery, and an 8F Swan-Ganz catheter was inserted via the right jugular vein into the pulmonary artery under pressure monitoring. Warmed sterile normal saline was infused at 0.2 ml·kg–1·min–1 iv via the Swan-Ganz catheter. A small right thoracotomy was made to allow a lung tissue sample to be obtained. After sheep were allowed to stabilize for 20 min, blood samples were taken for arterial blood gas analysis and leukocyte counts; cardiac output, pulmonary arterial pressure, pulmonary arterial wedge pressure, systemic arterial pressure, body temperature, and pulmonary vascular resistance were also recorded. These measurements were repeated until the sheep were at a steady state.

After steady state was established, a loading dose of 20 mg of I{kappa}B{alpha}-({Delta}N)-MTS was infused intravenously over 30 min. The upper chest wall was extirpated, and the diaphragm was incised to allow repeated biopsies of the lung. All of the above-listed measurements were repeated. One hour after I{kappa}B{alpha}-({Delta}N)-MTS infusion, LPS was infused intravenously at 2 µg/kg over 15 min. Measurements were made at 30-min intervals, and lung biopsies were taken before and 120 min after infusing endotoxin.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Production of recombinant cell-permeable proteins. A dominant inhibitory I{kappa}B{alpha} molecule was created lacking the sequences required for signal-dependent degradation [I{kappa}B{alpha}-({Delta}N)]. To produce the permeable fusion protein, the truncated cDNA of the I{kappa}B{alpha} molecule was cloned into an MTS vector that allows expression of the fusion protein tagged with GST on the NH2 terminus of the protein and the MTS motif on the COOH terminus of the protein (35). Recombinant cell-permeable GST-MTS, I{kappa}B{alpha}-({Delta}N)-MTS, and nonpermeable proteins were purified by a single-step chromatography using a glutathione agarose bead column. Endotoxin was removed from protein solutions using a Triton X-114 extraction procedure until endotoxin was reduced to acceptable levels.

To demonstrate that the recombinant proteins maintain their antigenicity, we subjected them to Western blot analysis using a specific antibody against the COOH-terminal portion of the I{kappa}B{alpha} molecule (Fig. 1A). Immunoblotting experiments detected the recombinant cell-permeable I{kappa}B{alpha}-({Delta}N) and nonpermeable proteins, indicating the preservation of antigenic epitopes during the production and purification steps of the recombinant proteins. The cell permeability of the I{kappa}B{alpha}-({Delta}N)-MTS was confirmed in NIH/3T3 fibroblast using an indirect immunofluorescent assay and analyzed by confocal microscopy. Cells treated with the permeable protein show an intracytoplasmic localization of the I{kappa}B{alpha}-({Delta}N)-MTS protein. No signal was detected in cells incubated with the nonpermeable protein (Fig. 1B). The amount of protein imported was directly dependent on the incubation time and the extracellular concentration of the permeable protein.



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  		Fig. 1. Shown are antigenicity and permeability of recombinant proteins I{kappa}B{alpha}-({Delta}N) and I{kappa}B{alpha}-({Delta}N)-MTS. A: recombinant nonpermeable I{kappa}B{alpha}-({Delta}N) and cell-permeable I{kappa}B{alpha}-({Delta}N)-MTS glutathione-S-transferase (GST) fusion proteins were induced with isopropylthiogalactoside (IPTG), purified, and subjected to Western blot analysis. The 2 recombinant proteins were recognized by an anti-I{kappa}B{alpha} antibody directed against a COOH-terminal motif of the protein. B: import assay of the I{kappa}B{alpha}-({Delta}N) and I{kappa}B{alpha}-({Delta}N)-MTS proteins in NIH/3T3 cells. Cells were incubated for 1 h with recombinant proteins, washed, and subjected to indirect immunofluorescent assay using an anti-GST antibody (red). Intracellular localization of the recombinant proteins was determined using a confocal fluorescent microscope. Cells exposed to the protein without the membrane translocating sequence (MTS) signal showed minimal background staining. Cells treated with the permeable protein I{kappa}B{alpha}-({Delta}N)-MTS show marked red staining that was localized primarily in the cytoplasm.

 
Inhibition of NF-{kappa}B activity in living cells. The I{kappa}B{alpha}-({Delta}N) molecule has been demonstrated to function as a repressor of multiple NF-{kappa}B proteins in transfection experiments and transgenic mice (6, 8, 26, 27, 29). To determine whether I{kappa}B{alpha}-({Delta}N)-MTS inhibits endogenous NF-{kappa}B/Rel signaling pathways in living cells, mobility shift analysis was performed with nuclear extracts from primary thymocytes treated with the recombinant proteins and activated with PMA and ionomycin. As shown in Fig. 2A, nuclear translocation of NF-{kappa}B complexes was detected only in cells without protein or in cells pretreated with the nonpermeable I{kappa}B{alpha}-({Delta}N) protein. Cells treated with the cell-permeable I{kappa}B{alpha}-({Delta}N)-MTS showed a dose-dependent reduction of the DNA/protein complex, suggesting sequestration of the complex in the cytoplasm. In addition, the observed inhibition of NF-{kappa}B pathway was selective in that NF-{kappa}B activity was intact in cells treated with the irrelevant cell-permeable protein GST-MTS (Fig. 2B). Similar data were obtained using Jurkat T cells (data not shown).



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  		Fig. 2. In vitro inhibition of NF-{kappa}B activation by I{kappa}B{alpha}-({Delta}N)-MTS is shown. A: primary thymocytes obtained from a young C57BL/6 mouse were incubated for 1 h with the indicated recombinant proteins and activated with PMA and ionomycin (Iono) for 30 min. Nuclear extracts were added to DNA binding mixtures containing a 32P-labeled {kappa}B probe. The major inducible complexes (p50-p65/cRel and p50-p50) are indicated with the arrows. Strong inhibition of NF-{kappa}B activity was observed with low and high concentrations of the permeable protein I{kappa}B{alpha}-({Delta}N)-MTS but not with I{kappa}B{alpha}-({Delta}N). B: specificity of I{kappa}B{alpha}-({Delta}N)-MTS-mediated inhibition of NF-{kappa}B pathway was tested in Jurkat T cells. Cells were treated with the control protein GST and GST-MTS and stimulated with PMA and ionomycin. Nuclear extracts were prepared and subjected to NF-{kappa}B EMSA. Comparable levels of NF-{kappa}B activation were detected after stimulation in cells without protein and treated with GST and GST-MTS proteins. C: inhibition of p65 and p50 nuclear translocation after activation in Jurkat T cells treated with I{kappa}B{alpha}-({Delta}N)-MTS protein. Jurkat T cells were incubated with the permeable proteins for 1 h and stimulated with PMA/ionomycin for 30 min. Cytoplasmic extracts were obtained, and p65 and p50 NF-{kappa}B subunits were detected by Western blotting. Levels of both NF-{kappa}B proteins decreased in the cytoplasm in stimulated cells treated without protein or I{kappa}B{alpha}-({Delta}N) protein. p65 And p50 proteins were retained in the cytoplasm in cells treated with the cell-permeable inhibitor I{kappa}B{alpha}-({Delta}N)-MTS.

 
The cytoplasmic sequestration of NF-{kappa}B proteins by I{kappa}B{alpha}-({Delta}N)-MTS was analyzed in Jurkat T cells. Cells were pretreated with permeable and nonpermeable I{kappa}B{alpha}-({Delta}N) and activated with PMA plus ionomycin. Cytoplasmic extracts were prepared and subjected to Western blot analyses using specific antibodies against p65 and p50 NF-{kappa}B subunits. Stimulated cells without protein and cells treated with nonpermeable protein showed a decrease in cytoplasmic concentration of p65 and p50 proteins after activation (Fig. 2C). In contrast, cells treated with the permeable inhibitor showed a constant amount of NF-{kappa}B/Rel proteins in the cytoplasm. These results demonstrate that the delivered permeable protein inhibited translocation of the NF-{kappa}B complex from cytoplasm to nucleus.

I{kappa}B{alpha}-({Delta}N)-MTS inhibits T cell expansion in activated primary T cells. Because transgenic inhibition of NF-{kappa}B pathways has been associated with defective T cell multiplication and in vivo clonal expansion, we determined the capacity of I{kappa}B{alpha}-({Delta}N)-MTS to regulate NF-{kappa}B-mediated responses by measuring T cell proliferation (6, 26). We used CD4+ T cells derived from DO11.10 TCR transgenic mice that respond specifically to the OVA323–339 peptide. DO11 transgenic T cells were preincubated with medium alone or I{kappa}B{alpha}-({Delta}N) and I{kappa}B{alpha}-({Delta}N)-MTS recombinant proteins and stimulated with several concentrations of OVA323–339 peptide in the presence of irradiated APC (34, 38, 41) (Fig. 3). After 48 h of stimulation, control cells treated without recombinant protein or the nonpermeable inhibitor I{kappa}B{alpha}-({Delta}N) showed a significant increase in the amount of DNA synthesized as determined by thymidine incorporation. Cells treated with the permeable I{kappa}B{alpha}-({Delta}N)-MTS showed decreased thymidine incorporation, indicating inhibition of the NF-{kappa}B-mediated proliferative signal. These data indicate that is possible to modify NF-{kappa}B-mediated responses in primary cells by the delivery of the I{kappa}B{alpha}-({Delta}N)-MTS.



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  		Fig. 3. Inhibition of T cell proliferation by the permeable inhibitor I{kappa}B{alpha}-({Delta}N)-MTS is depicted. Purified transgenic DO11.10 CD4+ T cells were incubated for 1 h with the purified fusion proteins, washed, and stimulated with the specific ovalbumin (OVA)323–339 peptide in the presence of antigen-presenting cells obtained from a wild-type BALB/c mouse. After 48 h of stimulation, DNA synthesis was measured by [3H]thymidine incorporation. Inhibition of T cell proliferation was exclusively observed in cells treated with I{kappa}B{alpha}-({Delta}N)-MTS protein. CPM, counts per minute.

 
In vivo inhibition of NF-{kappa}B-driven gene expression. To study in vivo the activity of the NF-{kappa}B-permeable inhibitor, we used the HLL transgenic mice that express in every tissue a luciferase reporter gene driven by the proximal HIV-LTR, an extensively characterized NF-{kappa}B-responsive promoter. Luciferase activity in these mice closely correlates with independent measurements of the NF-{kappa}B activation pathway and NF-{kappa}B-dependent gene expression (4, 5, 40). To induce local NF-{kappa}B activation in the skin, we made two 1.5-cm-long incisions through the skin and dermis. We imaged mice by bioluminescence and demonstrated that these incisions caused local NF-{kappa}B-luciferase expression in the HLL mice limited to the site of the injury. To determine whether I{kappa}B{alpha}-({Delta}N)-MTS would inhibit this NF-{kappa}B activation, we injected the recombinant protein intraperitoneally 2 and 12 h after surgery. Twenty-four hours after the last dose, luciferase substrate was injected intravenously (0.05 mg in 100 µl of PBS), and light emission was detected using a photon camera. NF-{kappa}B-dependent bioluminescence was detected only in animals that did not receive the permeable inhibitor protein after 1 and 3 min of exposure (Fig. 4A). After 5 min, the amount of light emitted by the animals that received the permeable form of the inhibitor was reduced 55% compared with the responses in animals that received control proteins (Fig. 4B). Because luciferase expression in this animal model is driven by NF-{kappa}B activity, these results indicate that systemic administration of I{kappa}B{alpha}-({Delta}N)-MTS was a competent inhibitor in vivo of the NF-{kappa}B pathway.



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  		Fig. 4. Systemic administration of the permeable inhibitor I{kappa}B{alpha}-({Delta}N)-MTS reduced local NF-{kappa}B activation in vivo. A: representative example of bioluminescence in a human immunodeficiency virus-long terminal repeat (HLL) mouse after skin wound. Mice with 2 incisions over the abdominal wall were treated with 2 doses of permeable I{kappa}B{alpha}-({Delta}N)-MTS protein or PBS. After 36 h of the skin wound, luciferin was administrated, and bioluminescence was measured 30 min later with a photon camera with 1, 3, and 5 min of exposure. Mice that received only PBS showed NF-{kappa}B activity at the site of the wound at all time points. In contrast, the I{kappa}B{alpha}-({Delta}N)-MTS-treated mouse had only limited NF-{kappa}B activation after 5 min of exposure. B: comparative analysis of bioluminescence in skin-wounded HLL mice after treatment with I{kappa}B{alpha}-({Delta}N) and I{kappa}B{alpha}-({Delta}N)-MTS proteins. Photon counts detected were substantially lower in mice receiving I{kappa}B{alpha}-({Delta}N)-MTS protein. Squares show the areas of the abdomen where photon counting was done.

 
Inhibition of responses of the lung to endotoxemia. To determine whether our inhibitor protein could prevent NF-{kappa}B activation in the lungs following endotoxemia and how that would alter the pathophysiology of the endotoxin response, we tested effects of intravenous administration of I{kappa}B{alpha}-({Delta}N)-MTS in a sheep preparation that we have studied extensively (7, 12, 48). Endotoxin is a known stimulus for NF-{kappa}B activation and altered lung physiology in sheep. To test the effect of I{kappa}B{alpha}-({Delta}N)-MTS, we treated sheep intravenously with 2 µg/kg of endotoxin alone or 1 h after intravenous administration of 20 mg of the recombinant protein.

Determination of nuclear translocation of p65 by immunohistochemistry analysis was used as a measurement of NF-{kappa}B activity after endotoxemia in lung samples taken 2 h after endotoxin treatment. As shown in Fig. 5A, endotoxin treatment induced nuclear localization of p65 as a consequence of NF-{kappa}B activation. In sharp contrast, samples from an I{kappa}B{alpha}-({Delta}N)-MTS-treated animal showed preferential localization of p65 in the cytoplasm, indicating inhibition of nuclear translocation (Fig. 5B). In addition, we analyzed NF-{kappa}B activity by mobility shift assays in lung samples collected during baseline and 2 h after administration of endotoxin. Consistent with the immunohistochemical analysis, endotoxemia resulted in activation of the NF-{kappa}B p65 subunit, and this activity was completely inhibited by prior administration of the I{kappa}B{alpha}-({Delta}N)-MTS protein. Interestingly, we observed an increase in activity of p50, the inhibitory subunit, which might provide additional inhibition of NF-{kappa}B-driven gene transcription (Fig. 5, C and D) (9).



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  		Fig. 5. Inhibition of LPS-induced NF-{kappa}B activation by in vivo administration of cell-permeable I{kappa}B{alpha}-({Delta}N)-MTS protein is shown. A: immunostaining for p65/NF-{kappa}B protein in lung samples from LPS-treated sheep showing nuclear localization of p65 (arrows). B: immunostaining for p65/NF-{kappa}B protein in lung samples from sheep treated with I{kappa}B{alpha}-({Delta}N)-MTS and LPS. Arrows indicate the predominant cytoplasmic localization of p65 subunit. C: NF-{kappa}B mobility shift analysis in lung whole cell extracts from sheep treated with LPS alone or with I{kappa}B{alpha}-({Delta}N)-MTS and LPS. I{kappa}B{alpha}-({Delta}N)-MTS administration before LPS treatment resulted in a striking decrease of p65 complexes. D: densitometry analysis corresponding to the p65 mobility shift in C.

 
We also determined pulmonary vascular resistance and arterial oxygenation 2 h after endotoxin administration in sheep that were pretreated with PBS or the I{kappa}B{alpha}-({Delta}N)-MTS protein. During the time of the administration of the protein, no changes were observed in physiological variables, suggesting that the protein was not toxic. In control animals, LPS administration caused a marked increase in the pulmonary vascular resistance and a marked decrease in arterial oxygenation. When the inhibitor protein was given before endotoxin, both of these physiological responses were attenuated (Fig. 6).



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  		Fig. 6. I{kappa}B{alpha}-({Delta}N)-MTS protein inhibits endotoxin pulmonary pathophysiology in sheep. A: pulmonary vascular resistance (PVR) was measured at baseline and 2 h after endotoxin treatment in sheep that previously received PBS or an infusion of I{kappa}B{alpha}-({Delta}N)-MTS protein. Administration of the NF-{kappa}B inhibitor resulted in 70% reduction in the endotoxin-induced increase in PVR. B: arterial oxygen tension (PO2) was measured in endotoxin and I{kappa}B{alpha}-({Delta}N)-MTS plus endotoxin-treated sheep. The endotoxin-mediated decrease of PO2 after 2 h of endotoxin was reduced 55% by I{kappa}B{alpha}-({Delta}N)-MTS protein.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Molecular therapies that target specific intracellular signaling pathways have potential as treatments of a range of diseases for which there is no effective therapy currently; this approach requires that the therapeutic agent access the cell interior. We and others have demonstrated that MTS fusion proteins are translocated with high efficiency into a large variety of cells, including endothelial and epithelial cells, by a process that depends on time, temperature, and protein concentration (35–37). This highly efficient delivery system can be used to study and manipulate various intracellular processes. For example, we created a cell-permeable form of the EGF-receptor binding protein by adding MTS to the binding protein Grb2-SH2 and used this fusion protein to study EGF-induced mitogenic pathways (37).

Several groups have confirmed the utility of the MTS technology. For instance, Zhao et al. (52) used an MTS system to deliver a functioning antibody into NIH/3T3 cells. Wang et al. (47), using MTS technology, showed that it is possible with a single immunization to enhance the CD4+ and CD8+ immune response against some specific tumors, resulting in complete inhibition of lung metastases and inducing protection from future tumor challenge. In addition, MTS technology has been used to deliver adaptor proteins, enzymes, transcription factors, and kinases, to clarify mechanisms of cell transformation, oncogenesis, and cardiac hypertrophy (45, 49)

We report here the development and in vivo delivery of a dominant negative inhibitor for NF-{kappa}B pathway using MTS technology. Expression of this I{kappa}B{alpha}-({Delta}N) inhibitor in T cells in transgenic mice demonstrated the critical role of NF-{kappa}B in T cell development and the regulation of T cell proliferative and apoptotic responses (6, 26, 27, 29). Additional studies using I{kappa}B{alpha}-({Delta}N) transgenic mice demonstrated that inhibition of NF-{kappa}B signal preferentially impairs type 1 compared with type 2 T helper-dependent responses. The defect in type 1 responses by inhibition of NF-{kappa}B has been associated with decreased incidence and severity of autoimmune disease in a model of collagen-induced arthritis in mice (42). Inappropriate and prolonged activation of NF-{kappa}B has been linked to several other diseases, including septic shock, acute respiratory distress syndrome, and ischemia-reperfusion injury. NF-{kappa}B has been also found to be constitutively activated and a common target and activator of oncogenes in cancer. Thus a membrane-permeable protein inhibitor of NF-{kappa}B could have considerable potential for application to human disease.

A developing strategy for inhibiting expression of specific proteins involves use of either antisense mRNA or small interfering RNA (43). Potential problems with this approach in vivo include possible less than desirable specificity and renal and hepatic toxicity. Efficacy may also depend on more efficient delivery and targeting systems (30).

Permeable protein inhibitors may offer advantages over nucleic acid-based therapeutics. Lin et al. (22) first described a protein containing a nuclear localization sequence that could block the trafficking of a variety of proteins from the cytosol into the nucleus. This peptide (SN50) blocks activation of NF-{kappa}B, but it is nonspecific; trafficking of other transcription factors to the nucleus (AP-1, STAT, N-FAT) is also inhibited (44). Another group of permeable inhibitors of NF-{kappa}B includes the cell-permeable inhibitor of NF-{kappa}B essential modulator (NEMO) (25). NEMO is one of the three subunits of the IKK complex. This permeable inhibitor is delivered using the TAT sequence derived from HIV, and it can block the classic NF-{kappa}B-activating pathways mediated by TNF-{alpha} (17) while maintaining intact alternative and atypical NF-{kappa}B activation pathways (CD40 and oxidative stress responses) (10, 18, 50).

Recent reports show that after the cells are activated, I{kappa}B and NF-{kappa}B proteins continuously traffic between the cytoplasm and the nucleus (32); a specific NF-{kappa}B inhibitor should have the capacity to interrupt this trafficking. I{kappa}B retains the NF-{kappa}B complex in the cytosol and can also turn off NF-{kappa}B activity by carrying NF-{kappa}B from the nucleus back into the cytosol. Thus approaches based on the I{kappa}B molecule have the potential to block classic and alternative NF-{kappa}B activation pathways with high specificity.

Our data suggest an early inhibition in the NF-{kappa}B activity by I{kappa}B{alpha}-({Delta}N)-MTS, suggesting the ability to displace the endogenous I{kappa}B{alpha} before activation; however, we cannot rule out the possibility that the inhibitor only binds to NF-{kappa}B complex after I{kappa}B{alpha} is degraded. We believe that the two events can occur simultaneously in cells treated with the inhibitor.

We demonstrate that I{kappa}B{alpha}-({Delta}N)-MTS readily accesses cell cytoplasm and shows good potency in inhibiting NF-{kappa}B activation in vitro and in vivo. Inhibition is effected by sequestering the NF-{kappa}B proteins into the cytoplasm even in the presence of an activating stimulus. Systemic administration of this inhibitor protein suppressed local NF-{kappa}B activation caused by wounding the skin in mice. In a sheep preparation, systemic administration of the inhibitor protein prevented endotoxemia-induced NF-{kappa}B activation in the lungs and moderated alterations in lung function caused by endotoxemia (40).

In conclusion, the dominant negative NF-{kappa}B inhibitor protein I{kappa}B{alpha}-({Delta}N) combined with MTS technology inhibits activation of NF-{kappa}B by sequestering those proteins in the cytoplasm even in the presence of potent activating stimuli. This is true both in cells in culture and in vivo. This technology could provide a new approach to developing therapeutics for the spectrum of diseases in which NF-{kappa}B activation is an important part of the pathogenesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Funding for this work was provided by Skin Diseases Research Core Center, Vanderbilt University School of Medicine, Emory University URC #2003100, National Heart, Lung, and Blood Institute Grants 5P01-HL-0669496-02 and 1RO1-HL-070891, and the McKelvey Center for Lung Transplantation.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge expert technical assistance of Smriti Bardhan, Nima Farsinejad, and Chris Cuppels, and we thank Dr. Jean-Francois Gauchat at University of Montreal for the nmLPS BL21 bacteria.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Rojas, Division of Pulmonary, Allergy, and Critical Care Medicine, Center for Translational Research of the Lung, Emory Univ. School of Medicine, Atlanta, GA 30322 (e-mail: mrojas{at}emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Baldwin AS Jr. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 14: 649–683, 1996.[CrossRef][ISI][Medline]
  2. Baldwin AS Jr. Series introduction: the transcription factor NF-{kappa}B and human disease. J Clin Invest 107: 3–6, 2001.[CrossRef][ISI][Medline]
  3. Ballard DW, Walker WH, Doerre S, Sista P, Molitor JA, Dixon EP, Peffer NJ, Hannink M, and Greene WC. The v-rel oncogene encodes a {kappa}B enhancer binding protein that inhibits NF-{kappa}B function. Cell 63: 803–814, 1990.[CrossRef][ISI][Medline]
  4. Blackwell TS, Yull FE, Chen CL, Venkatakrishnan A, Blackwell TR, Hicks DJ, Lancaster LH, Christman JW, and Kerr LD. Multiorgan nuclear factor {kappa}B activation in a transgenic mouse model of systemic inflammation. Am J Respir Crit Care Med 162: 1095–1101, 2000.[Abstract/Free Full Text]
  5. Blackwell TS, Yull FE, Chen CL, Venkatakrishnan A, Blackwell TR, Hicks DJ, Lancaster LH, Christman JW, and Kerr LD. Use of genetically altered mice to investigate the role of nuclear factor-{kappa}B activation and cytokine gene expression in sepsis-induced ARDS. Chest 116: 73S–74S, 1999.[Medline]
  6. Boothby MR, Mora AL, Scherer DC, Brockman JA, and Ballard DW. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-{kappa}B. J Exp Med 185: 1897–1907, 1997.[Abstract/Free Full Text]
  7. Brigham KL, Serafin W, Zadoff A, Blair I, Meyrick B, and Oates JA. Prostaglandin E2 attenuation of sheep lung responses to endotoxin. J Appl Physiol 64: 2568–2574, 1988.[Abstract/Free Full Text]
  8. Brockman JA, Scherer DC, McKinsey TA, Hall SM, Qi X, Lee WY, and Ballard DW. Coupling of a signal response domain in I{kappa}B{alpha} to multiple pathways for NF-{kappa}B activation. Mol Cell Biol 15: 2809–2818, 1995.[Abstract]
  9. Campbell IK, Gerondakis S, O’Donnell K, and Wicks IP. Distinct roles for the NF-{kappa}B1 (p50) and c-Rel transcription factors in inflammatory arthritis. J Clin Invest 105: 1799–1806, 2000.[ISI][Medline]
  10. Coope HJ, Atkinson PG, Huhse B, Belich M, Janzen J, Holman MJ, Klaus GG, Johnston LH, and Ley SC. CD40 regulates the processing of NF-{kappa}B2 p100 to p52. EMBO J 21: 5375–5385, 2002.[CrossRef][ISI][Medline]
  11. Du C, Yao S, Rojas M, and Lin YZ. Conformational and topological requirements of cell-permeable peptide function. J Pept Res 51: 235–243, 1998.[ISI][Medline]
  12. Duke SS, Guerry-Force ML, Forbes JT, Stewart SJ, King G, Wickersham N, Brigham KL, and Meyrick B. Acute endotoxin-induced lymphocyte subset sequestration in sheep lungs. Lab Invest 62: 355–362, 1990.[ISI][Medline]
  13. Epinat JC and Gilmore TD. Diverse agents act at multiple levels to inhibit the Rel/NF-{kappa}B signal transduction pathway. Oncogene 18: 6896–6909, 1999.[CrossRef][ISI][Medline]
  14. Fernandez T and Bayley H. Ferrying proteins to the other side. Nat Biotechnol 16: 418–420, 1998.[CrossRef][ISI][Medline]
  15. Finco TS and Baldwin AS. Mechanistic aspects of NF-{kappa}B regulation: the emerging role of phosphorylation and proteolysis. Immunity 3: 263–272, 1995.[CrossRef][ISI][Medline]
  16. Haskill S, Beg AA, Tompkins SM, Morris JS, Yurochko AD, Sampson-Johannes A, Mondal K, Ralph P, and Baldwin AS Jr. Characterization of an immediate-early gene induced in adherent monocytes that encodes I{kappa}B-like activity. Cell 65: 1281–1289, 1991.[CrossRef][ISI][Medline]
  17. Hsu H, Xiong J, and Goeddel DV. The TNF receptor 1-associated protein TRADD signals cell death and NF-{kappa}B activation. Cell 81: 495–504, 1995.[CrossRef][ISI][Medline]
  18. Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB, Mueller-Dieckmann C, Farahifar D, Rossi B, Auberger P, Baeuerle PA, and Peyron JF. Tyrosine phosphorylation of I{kappa}B-{alpha} activates NF-{kappa}B without proteolytic degradation of I{kappa}B-{alpha}. Cell 86: 787–798, 1996.[CrossRef][ISI][Medline]
  19. Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J, and Ruley HE. Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. Nat Biotechnol 19: 929–933, 2001.[CrossRef][ISI][Medline]
  20. Karin M. How NF-{kappa}B is activated: the role of the I{kappa}B kinase (IKK) complex. Oncogene 18: 6867–6874, 1999.[CrossRef][ISI][Medline]
  21. Lin YZ, Yao SY, and Hawiger J. Role of the nuclear localization sequence in fibroblast growth factor-1-stimulated mitogenic pathways. J Biol Chem 271: 5305–5308, 1996.[Abstract/Free Full Text]
  22. Lin YZ, Yao SY, Veach RA, Torgerson TR, and Hawiger J. Inhibition of nuclear translocation of transcription factor NF-B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem 270: 14255–14258, 1995.[Abstract/Free Full Text]
  23. Liu KY, Timmons S, Lin YZ, and Hawiger J. Identification of a functionally important sequence in the cytoplasmic tail of integrin {beta}3 by using cell-permeable peptide analogs. Proc Natl Acad Sci USA 93: 11819–11824, 1996.[Abstract/Free Full Text]
  24. Liu S, Tobias R, McClure S, Styba G, Shi Q, and Jackowski G. Removal of endotoxin from recombinant protein preparations. Clin Biochem 30: 455–463, 1997.[CrossRef][ISI][Medline]
  25. May MJ, D’Acquisto F, Madge LA, Glockner J, Pober JS, and Ghosh S. Selective inhibition of NF-{kappa}B activation by a peptide that blocks the interaction of NEMO with the I{kappa}B kinase complex. Science 289: 1550–1554, 2000.[Abstract/Free Full Text]
  26. Mora A, Youn J, Keegan A, and Boothby M. NF-{kappa}B/Rel participation in the lymphokine-dependent proliferation of T lymphoid cells. J Immunol 166: 2218–2227, 2001.[Abstract/Free Full Text]
  27. Mora AL, Chen D, Boothby M, and Rubin DH. Lineage-specific differences among CD8+ T cells in their dependence of NF-{kappa}B/Rel signaling. Eur J Immunol 29: 2968–2980, 1999.[CrossRef][ISI][Medline]
  28. Mora AL, Corn RA, Stanic AK, Goenka S, Aronica M, Stanley S, Ballard DW, Joyce S, and Boothby M. Antiapoptotic function of NF-{kappa}B in T lymphocytes is influenced by their differentiation status: roles of Fas, c-FLIP, and Bcl-xL. Cell Death Differ 10: 1032–1044, 2003.[CrossRef][ISI][Medline]
  29. Mora AL, Stanley S, Armistead W, Chan AC, and Boothby M. Inefficient ZAP-70 phosphorylation and decreased thymic selection in vivo result from inhibition of NF-{kappa}B/Rel. J Immunol 167: 5628–5635, 2001.[Abstract/Free Full Text]
  30. Morishita R, Tomita N, Kaneda Y, and Ogihara T. Molecular therapy to inhibit NF-{kappa}B activation by transcription factor decoy oligonucleotides. Curr Opin Pharmacol 4: 139–146, 2004.[CrossRef][ISI][Medline]
  31. Murphy KM, Heimberger AB, and Loh DY. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250: 1720–1723, 1990.[Abstract/Free Full Text]
  32. Nelson DE, Ihekwaba AE, Elliott M, Johnson JR, Gibney CA, Foreman BE, Nelson G, See V, Horton CA, Spiller DG, Edwards SW, McDowell HP, Unitt JF, Sullivan E, Grimley R, Benson N, Broomhead D, Kell DB, and White MR. Oscillations in NF-{kappa}B signaling control the dynamics of gene expression. Science 306: 704–708, 2004.[Abstract/Free Full Text]
  33. Pahl HL. Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 18: 6853–6866, 1999.[CrossRef][ISI][Medline]
  34. Robertson JM, Jensen PE and Evavold BD. DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323–339 epitope. J Immunol 164: 4706–4712, 2000.[Abstract/Free Full Text]
  35. Rojas M, Donahue JP, Tan Z, and Lin YZ. Genetic engineering of proteins with cell membrane permeability. Nat Biotechnol 16: 370–375, 1998.[CrossRef][ISI][Medline]
  36. Rojas M, Yao S, Donahue JP, and Lin YZ. An alternative to phosphotyrosine-containing motifs for binding to an SH2 domain. Biochem Biophys Res Commun 234: 675–680, 1997.[CrossRef][ISI][Medline]
  37. Rojas M, Yao S, and Lin YZ. Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor. J Biol Chem 271: 27456–27461, 1996.[Abstract/Free Full Text]
  38. Ross TM, Narayan M, Fang ZY, Minella AC, and Green PL. Human T-cell leukemia virus type 2 tax mutants that selectively abrogate NF-{kappa}B or CREB/ATF activation fail to transform primary human T cells. J Virol 74: 2655–2662, 2000.[Abstract/Free Full Text]
  39. Sadikot RT, Jansen ED, Blackwell TR, Zoia O, Yull F, Christman JW, and Blackwell TS. High-dose dexamethasone accentuates nuclear factor-{kappa}B activation in endotoxin-treated mice. Am J Respir Crit Care Med 164: 873–878, 2001.[Abstract/Free Full Text]
  40. Sadikot RT, Zeng H, Yull FE, Li B, Cheng DS, Kernodle DS, Jansen ED, Contag CH, Segal BH, Holland SM, Blackwell TS, and Christman JW. p47(Phox) deficiency impairs NF-{kappa}B activation and host defense in Pseudomonas pneumonia. J Immunol 172: 1801–1808, 2004.[Abstract/Free Full Text]
  41. Saparov A, Kraus LA, Cong Y, Marwill J, Xu XY, Elson CO, and Weaver CT. Memory/effector T cells in TCR transgenic mice develop via recognition of enteric antigens by a second, endogenous TCR. Int Immunol 11: 1253–1264, 1999.[Abstract/Free Full Text]
  42. Seetharaman R, Mora AL, Nabozny G, Boothby M, and Chen J. Essential role of T cell NF-{kappa}B activation in collagen-induced arthritis. J Immunol 163: 1577–1583, 1999.[Abstract/Free Full Text]
  43. Tomita N, Morishita R, Lan HY, Yamamoto K, Hashizume M, Notake M, Toyosawa K, Fujitani B, Mu W, Nikolic-Paterson DJ, Atkins RC, Kaneda Y, Higaki J, and Ogihara T. In vivo administration of a nuclear transcription factor-{kappa}B decoy suppresses experimental crescentic glomerulonephritis. J Am Soc Nephrol 11: 1244–1252, 2000.[Abstract/Free Full Text]
  44. Torgerson TR, Colosia AD, Donahue JP, Lin YZ, and Hawiger J. Regulation of NF-{kappa}B, AP-1, NFAT, and STAT1 nuclear import in T lymphocytes by noninvasive delivery of peptide carrying the nuclear localization sequence of NF-{kappa}B p50. J Immunol 161: 6084–6092, 1998.[Abstract/Free Full Text]
  45. Turkson J, Ryan D, Kim JS, Zhang Y, Chen Z, Haura E, Laudano A, Sebti S, Hamilton AD, and Jove R. Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation. J Biol Chem 276: 45443–45455, 2001.[Abstract/Free Full Text]
  46. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, and Miyamoto S. Rel/NF-{kappa}B/I{kappa}B family: intimate tales of association and dissociation. Genes Dev 9: 2723–2735, 1995.[Free Full Text]
  47. Wang S, Bartido S, Yang G, Qin J, Moroi Y, Panageas KS, Lewis JJ, and Houghton AN. A role for a melanosome transport signal in accessing the MHC class II presentation pathway and in eliciting CD4+ T cell responses. J Immunol 163: 5820–5826, 1999.[Abstract/Free Full Text]
  48. Wheeler AP, Jesmok G, and Brigham KL. Tumor necrosis factor’s effects on lung mechanics, gas exchange, and airway reactivity in sheep. J Appl Physiol 68: 2542–2549, 1990.[Abstract/Free Full Text]
  49. Wu Y, Temple J, Zhang R, Dzhura I, Zhang W, Trimble R, Roden DM, Passier R, Olson EN, Colbran RJ, and Anderson ME. Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation 106: 1288–1293, 2002.[Abstract/Free Full Text]
  50. Xiao G, Harhaj EW, and Sun SC. NF-{kappa}B-inducing kinase regulates the processing of NF-{kappa}B2 p100. Mol Cell 7: 401–409, 2001.[CrossRef][ISI][Medline]
  51. Yamamoto Y and Gaynor RB. Therapeutic potential of inhibition of the NF-{kappa}B pathway in the treatment of inflammation and cancer. J Clin Invest 107: 135–142, 2001.[ISI][Medline]
  52. Zhao Y, Lou D, Burkett J, and Kohler H. Chemical engineering of cell penetrating antibodies. J Immunol Methods 254: 137–145, 2001.[CrossRef][ISI][Medline]




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