From the
To control agonist-induced nuclear translocation of
transcription factor
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)
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-
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-
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-
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-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)(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
(I
B
)(6, 7, 8) . When cells are
activated with proinflammatory stimuli such as cytokines and
lipopolysaccharide (LPS), I
B
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) .
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.
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
Tyrosine-containing SKP and KP peptides
were radiolabeled with I-labeled Peptides
into NIH 3T3 Cells
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
10
M 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 I
B
in cytoplasmic
extract was similarly analyzed using human I
B
-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.
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-
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
Complexes in Agonist-stimulated Cells by the Cell-permeable Peptide
Carrying the Nuclear Localization Sequence (NLS)
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 I
B
in
agonist-stimulated THP-1 cells, albeit a trace amount of undegraded
I
B
was observed (Fig. 4D). Thus, these results
suggest that the major effect of SN50 peptide did not involve
proteolysis of I
B
.
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
I
B
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 I
B
stimulated by LPS (10). Immunoblot of equivalent amounts of cytosolic
extracts was obtained with anti-I
B
antibody.
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.
(
)
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.
B
, 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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.