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
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ABSTRACT |
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drug delivery; therapy; cell-permeable protein; inflammation; lung injury
This family of transcription factors includes p50, p52, c-Rel, RelB, and p65 (Rel A). In a resting state, NF-B proteins are sequestered in the cytoplasm as homo- or heterodimer (usually the heterodimer p50/p65) by association with I
B inhibitory proteins (I
B
, I
B
, and I
B
) (1, 46). I
B
, the best-characterized I
B molecule, contains serines at positions 32 and 36 that are phosphorylated by the activation complex of I
B kinases (IKK), separated from NF-
B, and then ubiquitinated, targeted to proteasomes, and degraded. Removal of I
B
from the complex exposes a nuclear localization sequence on NF-
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-B activity (13, 33, 51). In theory, NF-
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
B proteins; or 3) blocking the translocation of NF-
B dimers into the nucleus by targeting the nuclear pore protein complex. Only strategies that target the I
B molecules are likely to be specific for the NF-
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, 2123, 3537). 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-B-mediated cellular responses, we created a fusion protein containing a modified I
B
protein [termed I
B
-(
N)] that cannot be phosphorylated and therefore functions as a dominant negative NF-
B inhibitor. We found that the permeable I
B
-(
N)-MTS fusion protein inhibited nuclear translocation of NF-
B complex in cell culture and in vivo and attenuated NF-
B-dependent physiological responses in vivo.
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MATERIALS AND METHODS |
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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. 8523, revised 1996). Procedures in mice were approved by the Institutional Animal Care and Use Committee of Vanderbilt University.
Design of IB
-(
N)-MTS fusion protein.
cDNA from Jurkat T cells was used as a template to amplify specific I
B
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 37317) of I
B
. 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
B
-(
N)-MTS (35). As a control, we also produced a permeable GST-MTS and the nonpermeable GST and GST-I
B
-(
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 2030% 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 IB
-(
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 IB
-(
N)-MTS 1 h before endotoxin (see Open-chest sheep preparation) were used to prepare cell extracts. Gel mobility shift assays of NF-
B/Rel proteins were performed as previously described using a double-stranded 32P-labeled oligonucleotide modified from
B enhancer sequences in the IL-2 receptor-
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 (OVA323339) (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. OVA323339 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-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 40 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·kg1·min1 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 IB
-(
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
B
-(
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.
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RESULTS |
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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 IB
molecule (Fig. 1A). Immunoblotting experiments detected the recombinant cell-permeable I
B
-(
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
B
-(
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
B
-(
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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IB
-(
N)-MTS inhibits T cell expansion in activated primary T cells.
Because transgenic inhibition of NF-
B pathways has been associated with defective T cell multiplication and in vivo clonal expansion, we determined the capacity of I
B
-(
N)-MTS to regulate NF-
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 OVA323339 peptide. DO11 transgenic T cells were preincubated with medium alone or I
B
-(
N) and I
B
-(
N)-MTS recombinant proteins and stimulated with several concentrations of OVA323339 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
B
-(
N) showed a significant increase in the amount of DNA synthesized as determined by thymidine incorporation. Cells treated with the permeable I
B
-(
N)-MTS showed decreased thymidine incorporation, indicating inhibition of the NF-
B-mediated proliferative signal. These data indicate that is possible to modify NF-
B-mediated responses in primary cells by the delivery of the I
B
-(
N)-MTS.
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Determination of nuclear translocation of p65 by immunohistochemistry analysis was used as a measurement of NF-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-
B activation. In sharp contrast, samples from an I
B
-(
N)-MTS-treated animal showed preferential localization of p65 in the cytoplasm, indicating inhibition of nuclear translocation (Fig. 5B). In addition, we analyzed NF-
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-
B p65 subunit, and this activity was completely inhibited by prior administration of the I
B
-(
N)-MTS protein. Interestingly, we observed an increase in activity of p50, the inhibitory subunit, which might provide additional inhibition of NF-
B-driven gene transcription (Fig. 5, C and D) (9).
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DISCUSSION |
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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-B pathway using MTS technology. Expression of this I
B
-(
N) inhibitor in T cells in transgenic mice demonstrated the critical role of NF-
B in T cell development and the regulation of T cell proliferative and apoptotic responses (6, 26, 27, 29). Additional studies using I
B
-(
N) transgenic mice demonstrated that inhibition of NF-
B signal preferentially impairs type 1 compared with type 2 T helper-dependent responses. The defect in type 1 responses by inhibition of NF-
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-
B has been linked to several other diseases, including septic shock, acute respiratory distress syndrome, and ischemia-reperfusion injury. NF-
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-
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-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-
B includes the cell-permeable inhibitor of NF-
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-
B-activating pathways mediated by TNF-
(17) while maintaining intact alternative and atypical NF-
B activation pathways (CD40 and oxidative stress responses) (10, 18, 50).
Recent reports show that after the cells are activated, IB and NF-
B proteins continuously traffic between the cytoplasm and the nucleus (32); a specific NF-
B inhibitor should have the capacity to interrupt this trafficking. I
B retains the NF-
B complex in the cytosol and can also turn off NF-
B activity by carrying NF-
B from the nucleus back into the cytosol. Thus approaches based on the I
B molecule have the potential to block classic and alternative NF-
B activation pathways with high specificity.
Our data suggest an early inhibition in the NF-B activity by I
B
-(
N)-MTS, suggesting the ability to displace the endogenous I
B
before activation; however, we cannot rule out the possibility that the inhibitor only binds to NF-
B complex after I
B
is degraded. We believe that the two events can occur simultaneously in cells treated with the inhibitor.
We demonstrate that IB
-(
N)-MTS readily accesses cell cytoplasm and shows good potency in inhibiting NF-
B activation in vitro and in vivo. Inhibition is effected by sequestering the NF-
B proteins into the cytoplasm even in the presence of an activating stimulus. Systemic administration of this inhibitor protein suppressed local NF-
B activation caused by wounding the skin in mice. In a sheep preparation, systemic administration of the inhibitor protein prevented endotoxemia-induced NF-
B activation in the lungs and moderated alterations in lung function caused by endotoxemia (40).
In conclusion, the dominant negative NF-B inhibitor protein I
B
-(
N) combined with MTS technology inhibits activation of NF-
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-
B activation is an important part of the pathogenesis.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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