©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Nuclear Translocation of Transcription Factor NF-B by a Synthetic Peptide Containing a Cell Membrane-permeable Motif and Nuclear Localization Sequence (*)

Yao-Zhong Lin (§) , SongYi Yao , Ruth Ann Veach , Troy R. Torgerson , Jacek Hawiger (§)

From the (1)Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To control agonist-induced nuclear translocation of transcription factor B (NF-B) in intact cells, cell-permeable synthetic peptides were devised. Their import into intact cells was dependent on a hydrophobic region selected from the signal peptide sequences and was verified by their inaccessibility to extracellular proteases and by confocal laser scanning microscopy. When a cell-permeable peptide carried a functional cargo representing the nuclear localization sequence of NF-B p50, it inhibited in a concentration-dependent manner nuclear translocation of NF-B in cultured endothelial and monocytic cells stimulated with lipopolysaccharide or tumor necrosis factor-. Synthetic peptide analogues with deleted hydrophobic cell membrane-permeable motif or with a mutated nuclear localization sequence were inactive. Cell membrane-permeable peptides were not cytotoxic within the concentration range used in these experiments. These results suggest that cell-permeable synthetic peptides carrying a functional cargo can be applied to control signal transduction-dependent subcellular traffic of transcription factors mediating the cellular responses to different agonists. Moreover, this approach can be used to study other intracellular processes involving proteins with functionally distinct domains.


INTRODUCTION

In response to the binding of extracellular ligands to cell surface receptors, multiple transcription factors are activated in the cytoplasm and translocated to the nucleus where they exert positive or negative control over cellular genes. Such subcellular traffic of transcription factors usually requires the presence of a positively charged nuclear localization sequence (NLS)()(1) . For example, this sequence is required for nuclear translocation of NF-B(2) , which plays a critical role in regulating a number of cellular and viral genes, including the enhancer of human immunodeficiency virus (HIV) (for review see Refs. 3-5). In unstimulated immune and nonimmune cells (preB cells, T cells, monocytic cells, and endothelial cells), NF-B remains in the cytoplasm in the inactive form composed of p50 (NFKB1), p65 (RelA), and an inhibitor B (IB)(6, 7, 8) . When cells are activated with proinflammatory stimuli such as cytokines and lipopolysaccharide (LPS), IB is degraded (for review see Ref. 5). This degradation in human monocytic cells unmasks the NLS of NF-B/Rel subunits, allowing the nuclear translocation of p50/p65 and p50/c-Rel complexes(9, 10) .

We have devised a new approach to investigate the role of the NLS in subcellular trafficking of transcription factors in intact cells. To deliver the functional domain (such as the NLS) of a selected protein from the outside to the inside of intact cells, we chose as a carrier the hydrophobic region (h-region) of the signal peptide(11) , a segment known to interact with lipid bilayers. When such a cell-permeable peptide carried a functional cargo in the form of the NLS of the transcription factor NF-B p50, it inhibited subcellular traffic of NF-B/Rel complexes from the cytoplasm to the nucleus in cultured endothelial and monocytic cells stimulated with proinflammatory agonists. This approach, called cell-permeable peptide import (CPPI), obviates the need for permeabilization with pore-forming reagents or microinjection of individual cells.


MATERIALS AND METHODS

Peptides and Antibodies

Peptides were synthesized by a stepwise solid-phase peptide synthesis method and purified by C reverse-phase HPLC on a Vydac column eluted with a linear gradient of 10-60% acetonitrile, 0.05% trifluoroacetic acid as described(12, 13) . The molecular weight of the purified peptides was verified by mass spectrometry analysis. Polyclonal anti-peptide antibody against the SM peptide conjugated to keyhole limpet hemocyanin was raised in rabbits, and IgG was purified by Protein A-Sepharose chromatography. This antibody recognized both SM and SN50 peptides through interaction with common epitopes such as the carboxyl-terminal sequence LMP present in both peptides (Fig. 1).


Figure 1: Sequences of cell membrane-permeable and control peptides (single-letter amino acid code). The membrane-translocating hydrophobic sequence derived from the h-region of the predicted signal peptide sequence of K-FGF is underlined. The peptides without this hydrophobic sequence (KP and N50) were used as comparative controls. The nuclear localization sequence of NF-B p50 (2) is printed in bold face, and the mutated residues in the SM and SN50M peptides are in italics.



Translocation of I-labeled Peptides into NIH 3T3 Cells

Tyrosine-containing SKP and KP peptides were radiolabeled with I by the IODOGEN method (Pierce). The specific activities of I-labeled SKP and KP peptides were 3.6 10 cpm/ng and 7.1 10 cpm/ng, respectively. NIH 3T3 cells grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) were subcultured on a 60-mm dish and incubated at 37 °C for 3 days. The confluent monolayers (1.6 10 cells) on each dish were then washed twice with phosphate-buffered saline (PBS) and treated with 15 ng of I-SKP or I-KP peptide at 37 °C for 30 min. The cells were washed eight times with PBS and twice with 2 M NaCl buffer (pH 7.5) until no radioactivity could be detected in the washings. The washed cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1% Triton X-100), and the radioactivity in the cell lysates was counted in a Packard Auto-Gamma counter. The mobility of I-SKP peptide in cell lysates was the same as that of free I-labeled SKP peptide on 15% SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Protease Treatment of Cells following Peptide Import

Confluent NIH 3T3 cells were incubated with I-SKP peptide and washed as described above. Subsequently, the washed cells were incubated with Pronase (1 mg/ml) or trypsin (0.5 mg/ml) in DMEM for 5 min at 37 °C. The cells were separated by centrifugation, and the radioactivity in the cell lysates and supernatant was counted separately. The ability of proteases to degrade free SKP peptide under the same conditions was established by analysis of untreated and protease-treated peptide on C reverse-phase HPLC.

Intracellular ATP Depletion Studies

Confluent NIH 3T3 cells were incubated with 5 µg/ml antimycin, 6.5 mM 2-deoxyglucose, and 10 mM glucono--lactone in DMEM for 2 h at 37 °C(14) . The ATP-depleted cells were then treated with I-SKP peptide and washed, and the cell-associated radioactivity was determined as described above. The levels of ATP in ATP-depleted and nondepleted cells were determined by an ATP bioluminescence assay kit (Sigma). ATP levels in ATP-depleted cells were reduced by 90-95% as compared to nondepleted cells.

Endosomal/Lysosomal Function and Protein Synthesis Inhibitor Studies

Confluent NIH 3T3 cells were incubated with ammonium chloride (50 mM), chloroquine (0.1 mM), cycloheximide (10 µg/ml), or diluent in DMEM for 1 h at 37 °C. The cells were then treated with I-SKP peptide and washed, and the cell-associated radioactivity was determined as described above.

Indirect Immunofluorescence Assay and Confocal Laser Scanning Microscopy Analysis

Confluent murine endothelial LE-II cell line (15) or NIH 3T3 cells grown on chamber slides (Nunc) were treated with 0.5 ml of peptide solution in DMEM containing 10% FBS under the conditions indicated in the figures. The cells were washed three times with cold PBS and then fixed with 3.5% paraformaldehyde solution in PBS at 4 °C for 20 min. The fixed cells were washed three times with cold PBS and treated with 0.25% Triton X-100 in PBS for 10 min. The washed cells were then incubated with anti-peptide IgG in PBS for 1 h. After three 5-min washings with PBS, the intracellular peptide-antibody complexes were subsequently detected with rhodamine-labeled goat anti-rabbit IgG (Kirkegaard & Perry) after a 1-h incubation. Coverslips with stained cells were mounted in Poly/Mount (Polyscience) and analyzed in a Leitz confocal laser scanning microscope system using a 100 oil immersion lens. The color images were stored in Tiff format and printed by ColorCopy, Nashville, TN.

Gel Mobility Shift Assay and Immunoblot Analysis

Confluent murine endothelial LE-II cells in DMEM containing 10% FBS or a suspension of human monocytic THP-1 cells (1 10 cells/ml) in RPMI 1640 medium supplemented with 50 units/ml penicillin, 50 µg/ml streptomycin, 5 10M 2-mercaptoethanol, and 10% FBS were treated with or without peptides at the indicated concentrations for 15 min at 37 °C. Cells were then further incubated with LPS (10 ng/ml), TNF (100 units/ml), or diluent for another 1 or 2 h. Equivalent amounts of nuclear extracts prepared from untreated or peptide-treated cells were assayed for B binding activity of NF-B/Rel with P-labeled double-stranded oligonucleotide B probe derived from the mouse immunoglobulin enhancer (and the HIV long terminal repeat) as described(9) . The composition of the B binding complexes was determined using monospecific antisera against p50, p65, and c-Rel added to nuclear extracts prior to electrophoretic mobility shift assay (EMSA)(9) . To verify the presence of translocated p50 subunit in the nucleus, nuclear extracts of THP-1 cells prepared as described above were electrophoresed on 8% SDS-polyacrylamide gels and transferred to Hybond-ECL. The blot was then analyzed for immunoreactive p50 protein with monospecific rabbit antiserum against the NH-terminal segment of p50/p105 followed by horseradish peroxidase-linked donkey anti-rabbit antibody according to ECL protocol as described(9) . Immunoreactive IB in cytoplasmic extract was similarly analyzed using human IB-specific (amino acids 289-317) antipeptide rabbit antibody(10) .

Cytotoxicity Assay

Freshly prepared fluorescein diacetate/ethidium bromide in PBS was added to the peptide-treated or untreated cells in culture dishes at room temperature for 5 min(16) . Cells were then observed by fluorescence microscope and orange-stained cells were counted as not viable.


RESULTS

Import of Synthetic Peptides into Cells Is Mediated by the Hydrophobic Sequence of the Signal Peptide

To design cell-permeable peptides capable of carrying a functional domain such as NLS into living cells, we selected the hydrophobic region (h-region) of the signal peptide sequence as the membrane-translocating carrier. We reasoned that this region, known to facilitate secretion of proteins (11), can be applied to import the synthetic peptides to the inside of a cell. Accordingly, we first synthesized a 41-residue peptide (referred to as SKP, see Fig. 1) comprising the h-region of the signal sequence of Kaposi fibroblast growth factor (K-FGF) (17) in its amino-terminal segment while the cleavage site for a signal peptidase was deleted. The carboxyl-terminal segment of this peptide contained residues 129-153 of K-FGF with tyrosine available for radiolabeling with I. To determine the import of SKP peptide into cells, NIH 3T3 cells were incubated with I-labeled SKP peptide and the amount of cell-associated I-SKP peptide was measured. In three independent experiments, 4% of the added I-SKP peptide was associated with the cells following a 30-min incubation. Resistance to the protease treatment serves as a criterion for membrane translocation rendering the transported peptide inaccessible to proteases in other systems such as membrane vesicles(18) . Accordingly, we treated the cells containing I-SKP peptide with Pronase and trypsin. No significant loss of cell-associated radioactivity was observed after protease treatment (untreated: 21,323 ± 853, Pronase-treated: 21,791 ± 1,953, and trypsin-treated: 23,193 ± 310 cpm/1 10 cells). Because both proteases readily digested SKP peptide in a cell-free system, it appears that cell-associated I-SKP peptide was inaccessible to extracellular proteases. On average, 20 times more (molar concentration) I-SKP peptide was associated with the cells compared to the control I-KP peptide lacking a hydrophobic region. This indicates that the h-region of the K-FGF signal sequence mediates the cellular import of SKP peptide.

I-SKP peptide import into cells was not blocked by adding a 200-fold excess of unlabeled SKP peptide, indicating the lack of receptor-mediated import of I-SKP peptide. Inhibitors of endosomal/lysosomal function, ammonium chloride and chloroquine, as well as the protein synthesis inhibitor, cycloheximide, did not affect cellular uptake of I-SKP peptide compared to control cells (23,040 ± 2,489, 26,138 ± 5,346, 25,671 ± 522, and 26,089 ± 3,174 cpm/1.6 10 cells, respectively). Intracellular ATP as a high energy source did not seem to be required for peptide import because I-SKP peptide import into cells depleted of ATP was similar to that of control cells (22,266 ± 3,602 versus 20,189 ± 2,109 cpm/1.6 10 cells).

Inhibition of the Nuclear Translocation of NF-B Complexes in Agonist-stimulated Cells by the Cell-permeable Peptide Carrying the Nuclear Localization Sequence (NLS)

Having demonstrated the feasibility of cell-permeable peptide import and its characteristics, we tested the hypothesis that such peptides can carry a functional domain such as the NLS. Therefore, we synthesized a cell-permeable peptide bearing the NLS of NF-B p50 subunit (SN50 in Fig. 1). We employed confocal laser scanning microscopy to verify the import of the SN50 peptide in murine endothelial LE-II cells by analyzing the fluorescent signal in an indirect immunofluorescence assay. A six-step Z-position sectional scanning demonstrated the strongest fluorescent signal representing immunoreactive SN50 peptide in the midsections (panels 3-5) of a cell (Fig. 2). Immunodetection of the peptide was specific because cells exposed to SN50 peptide followed by incubation with peptide-antibody complex or with secondary antibody alone (omitting primary antibody) did not have a fluorescence signal. These results indicate that the gain in fluorescence in the SN50 peptide-treated LE-II cells (Fig. 2) was due to the cellular import of SN50 peptide. This import was time-, concentration-, and temperature-dependent. At 4 °C, no import of cell-permeable peptide was observed. The maximal fluorescence was observed at 37 °C between 30 min and 1 h in the concentration range of 50 to 100 µg/ml. Within this concentration range, the peptide was not cytotoxic, as determined by fluorescein diacetate/ethidium bromide staining of peptide-treated cultured cells (16).


Figure 2: Demonstration of the intracellular localization of SN50 peptide in LE-II cells by confocal laser scanning microscopy analysis. Confluent LE-II cells were treated at 37 °C with 50 µg/ml SN50 peptide for 30 min. The intracellular peptide was detected as yellow stains by an indirect immunofluorescence assay using anti-SM peptide IgG and rhodamine-labeled anti-rabbit antibody and analyzed by a six-step Z-position sectional scanning of the cell. Panels 1-7, 1-µm cell sections from the bottom (panel 1) to the top (panel 7) of a representative SN50 peptide-treated cell. Panel 8, the composite image composed of all seven sections of the SN50 peptide-treated cell. Panel 9, the composite image of the untreated cell.



The functional effect of the SN50 peptide on nuclear translocation of NF-B/Rel complexes in cells activated by the proinflammatory agonists, LPS and TNF, was determined in an electrophoretic mobility shift assay (EMSA). As shown in Fig. 3A, LPS-induced nuclear translocation of the NF-B complexes in LE-II cells was specifically inhibited by SN50 peptide. In contrast, three control peptides, N50, SM, and SN50M, were without measurable effect (Fig. 3A and data not shown). N50 peptide lacks a cell-permeable hydrophobic region and contains only the NLS of p50. Cell-permeable SM and SN50M peptides have mutations in seven and two of ten residues within the NLS of SN50 peptide, respectively (Fig. 1). The inhibition of NF-B nuclear translocation by SN50 peptide was concentration-dependent, reaching maximum at the extracellular concentration of 50 µg/ml (18 µM) (Fig. 3B). To exclude the possibility that the inhibition by SN50 peptide was caused by its interference with the binding of oligonucleotide probe to the NF-B complex, SN50 peptide was incubated in vitro with nuclear extracts and a radiolabeled probe. B binding activity remained unchanged in nuclear extracts from LPS-stimulated cells (data not shown). Therefore, the observed inhibitory effect of SN50 reflects its ability to enter the cell and to compete with NF-B complexes for the cellular machinery responsible for nuclear translocation of NF-B in murine endothelial cell lines.


Figure 3: A, cell-permeable SN50 peptide inhibits nuclear translocation of NF-B induced by LPS in murine endothelial LE-II cell line. Confluent LE-II cells were treated with different peptides (50 µg/ml) for 15 min prior to stimulation with LPS (10 ng/ml) for 2 h. Equivalent amounts of nuclear extracts were prepared and assayed for B binding activity with P-labeled double-stranded oligonucleotide B probe using electrophoretic mobility shift assay (EMSA). B, inhibitory effect of SN50 peptide on nuclear translocation of NF-B in LE-II cells is concentration-dependent. Confluent LE-II cells were treated with SN50 peptide at the indicated concentrations for 15 min and then with LPS (10 ng/ml) for 1 h. Nuclear extracts were assayed using EMSA. The phosphorimage analysis of the gel before autoradiography indicates that inhibition by SN50 peptide was 13% at 10 µg/ml, 46% at 30 µg/ml, and 88% at 50 µg/ml.



The SN50 peptide was also active in the human monocytic THP-1 cell line. The LPS- or TNF-induced nuclear translocation of NF-B was inhibited in SN50 peptide-treated THP-1 cells as demonstrated in EMSA (Fig. 4A). As exemplified by LPS-stimulated cells, inhibition by SN50 was concentration-dependent (Fig. 4B). Again, three control peptides, nonpermeable N50 peptide, cell-permeable SM, and SN50M peptides, did not prevent nuclear translocation of NF-B in this cell line although SN50M peptide showed partial inhibition (Fig. 4A). Consistent with the results of the EMSA, the immunoblot analysis of the nuclear extracts of LPS- or TNF-stimulated THP-1 cells pretreated with SN50 peptide did not show detectable p50 protein (Fig. 4C). Addition of SN50 peptide (100 µg/ml) did not prevent the intracellular degradation of IB in agonist-stimulated THP-1 cells, albeit a trace amount of undegraded IB was observed (Fig. 4D). Thus, these results suggest that the major effect of SN50 peptide did not involve proteolysis of IB.


Figure 4: A, cell-permeable SN50 peptide inhibits nuclear translocation of NF-B induced by TNF or LPS in human monocytic THP-1 cell line. THP-1 cells (1 10 cells/ml) were treated with different peptides (100 µg/ml) for 15 min prior to stimulation with LPS (10 ng/ml) for 2 h or TNF (100 units/ml) for 1 h. Equivalent amounts of nuclear extracts were prepared and assayed for B binding activity using EMSA. B, inhibitory effect of SN50 peptide on nuclear translocation of NF-B in THP-1 cells is concentration-dependent. THP-1 cells were treated with SN50 peptide at the indicated concentrations for 15 min and then with LPS (10 ng/ml) for 2 h. Nuclear extracts were assayed using EMSA. Inhibition by SN50 peptide was 27% at 10 µg/ml, 49% at 30 µg/ml, 66% at 50 µg/ml, and 85% at 100 µg/ml according to the phosphorimage analysis . C, immunoblot analysis of NF-B p50 in nuclear extracts of agonist-stimulated THP-1 cells. THP-1 cells were treated with or without SN50 peptide (100 µg/ml) and TNF (100 units/ml) or LPS (10 ng/ml) as described above. Immunoblot of equivalent amounts of nuclear extracts was obtained with anti-NF-B p50 antibody. D, immunoblot analysis of IB in cytosolic extracts of LPS-stimulated THP-1 cells. THP-1 cells were treated with or without peptides (100 µg/ml) and LPS (10 ng/ml) as described above. Cells were pretreated with cycloheximide (10 µg/ml) to inhibit de novo synthesis of IB stimulated by LPS (10). Immunoblot of equivalent amounts of cytosolic extracts was obtained with anti-IB antibody.




DISCUSSION

Thus far, intracellular protein trafficking and protein-protein interactions involved in signal transduction and gene transcription have been analyzed largely through the use of mutated proteins expressed in cells transfected with appropriate constructs (2) or through the use of inhibitory molecules introduced after permeabilization of cell membranes with pore-forming reagents or microinjection of individual cells(19) . In contrast, the CPPI technique described in this report allows easy delivery of the nuclear localization sequence of NF-B into intact cells to influence agonist-induced nuclear translocation of NF-B/Rel complexes. These complexes translocated to the nucleus are made of p50/p65 and p50/p50 dimers in LE-II cells (this study) and p50/p65 and p50/c-Rel dimers in THP-1 cells(9) . Therefore, inhibition of the nuclear translocation of NF-B in both cell types by cell-permeable SN50 peptide indicates that this peptide can block the intracellular recognition mechanism for the NLS present on at least three members of NF-B/Rel family. This inhibition is specific for the NLS sequence of SN50 peptide because cell-permeable peptide analogues SM and SN50M with a mutated NLS sequence did not prevent nuclear translocation of NF-B/Rel complexes. However, the cell-permeable peptide containing the NLS from SV40 virus large T antigen inhibited nuclear translocation of the NF-kB (data not shown). This result is not surprising because other studies have suggested that various nuclear proteins containing different NLSs may share similar nuclear import mechanisms (for recent review see Ref. 20). The inhibitory effect demonstrated in both LPS- and TNF-treated THP-1 cells suggests that irrespective of the agonists used, the nuclear translocation machinery is equally sensitive to the inhibitory effect of SN50 peptide. Consistent with these results, SN50 peptide inhibited the LPS-induced reporter gene activity measured by a chloramphenicol acetyltransferase assay in a transiently transfected endothelial LE-II cell line.()Thus, inhibiting the subcellular traffic of NF-B in vivo may prove feasible for suppressing the cellular response to agonists inducing NF-B-dependent gene transcription.

The ideal characteristics for cell-permeable peptides carrying a functional cargo include cell membrane permeability without cytotoxic effects, functional selectivity, and easy detectability. The peptides we designed fulfilled these criteria. In addition, the plasma membranes of different cell types were permeable to the synthetic peptides used in our study. Although we used only the hydrophobic region derived from the signal sequence of K-FGF in this study, hydrophobic regions from other signal sequences also endowed peptides with cell membrane permeability.()

Until now, signal peptides have been known to facilitate export of many secretory proteins across eukaryotic endoplasmic reticulum or bacterial cytoplasmic membranes through a hydrophilic protein-conducting channel (for review see Refs. 21 and 22). However, the ATP-independent mechanism of CPPI designed by us seems to be different. It appears that cell membrane fluidity and membrane protein mobility are important for CPPI because low temperature or pretreatment of cells with paraformaldehyde prevents this process. However, receptor-mediated uptake does not seem to be involved. This is because an excess of unlabeled peptide, the inhibitors of endosomal/lysosomal uptake, and an inhibitor of protein synthesis (23) were without effect.

Taken together, our results document that the cell-permeable SN50 peptide carrying a functional domain, NLS, can inhibit nuclear translocation of NF-B/Rel complexes in intact cells. It is plausible that other intracellular processes involving proteins with functionally distinct domains can be probed using the same approach.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants HL45994 and HL30647 and a Mellon Foundation Award for Faculty Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 615-343-8277 or 615-343-8280; Fax: 615-343-7392.

The abbreviations used are: NLS, nuclear localization sequence; CPPI, cell-permeable peptide import; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; PBS, phosphate-buffered saline; IB, inhibitor B; K-FGF, Kaposi fibroblast growth factor; LPS, lipopolysaccharide; NF-B, nuclear factor B; TNF, tumor necrosis factor-; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium.

A. Burzynski and J. Hawiger, unpublished results.

X.-Y. Liu, S. Timmons, Y.-Z. Lin, and J. Hawiger, unpublished results.


ACKNOWLEDGEMENTS

We thank F. F. Ebner, L. Franklin, and M. J. Maguire for their help with confocal microscopy, D. W. Ballard, W. C. Greene, and A. Isral for the NF-B antibodies, T. Maciag for the LE-II cell line, C. Walter for editorial assistance, and G. N. Green, J. G. Hardman, R. E. Ruley, and J. P. Tam for review of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.