1 School of Biological Sciences, University of Liverpool, Crown Street,
Liverpool, L69 7ZB, UK
2 AstraZeneca R&D Charnwood, Molecular Biology, Bakewell Road, Loughborough,
Leicestershire, LE11 5RH, UK
3 Kinetic Imaging Ltd, 2 Brunel Road, Wirral, CH62 3NY, UK
* Author for correspondence (e-mail: mwhite{at}liv.ac.uk )
Accepted 13 December 2001
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Summary |
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When cells were treated with the CRM-1-dependent nuclear export inhibitor,
leptomycin B (LMB), there was nuclear accumulation of IB
-EGFP
and p65-dsRed, with I
B
-EGFP accumulating more rapidly. No
NF-
B-dependent transcriptional activation was seen in response to LMB
treatment. Following 1 hour treatment with LMB, significant
I
B
-EGFP nuclear accumulation, but low levels of p65-dsRed
nuclear accumulation, was observed. When these cells were stimulated with
TNF
, degradation of I
B
-EGFP was observed in both the
cytoplasm and nucleus. A normal transient transcription response was observed
in the same cells using luminescence imaging of NF-
B-dependent
transcription. These observations suggest that both normal activation and
post-induction repression of NF-
B-dependent transcription occur even
when nuclear export of NF-
B is inhibited. The results provide
functional evidence that other factors, such as modification of p65 by
phosphorylation, or interaction with other proteins such as transcriptional
co-activators/co-repressors, may critically modulate the kinetics of
transcription through this signalling pathway.
Key words: NF-B, Signal transduction, Fluorescent protein fusions, Firefly luciferase, Luminescence imaging, Confocal microscopy
![]() |
Introduction |
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TNF-induced activation of NF-
B involves stimulation of the
TNF
receptor 1 (TNFR1) through binding of TNF
, which induces
trimerisation of TNFR1. This is followed by recruitment of cytosolic factors
including TNF receptor-associated death domain, TRADD
(Hsu et al., 1995
) and TNF
receptor associated factors (TRAFs)
(Pomerantz and Baltimore,
1999
). This leads to stimulation of a signalling pathway that acts
through a multiprotein kinase complex called the signalsome, which is known to
include NF-
B-inducing kinase (NIK) and I
B kinases IKK
,
IKKß (Zandi et al., 1997
;
DiDonato et al., 1997
;
Malinin et al., 1997
;
Mercurio et al., 1997
;
Woronicz et al., 1997
), and
IKKi in immune cells (Shimada
et al., 1999
). Two scaffold proteins, IKK
and IKK complex
associated protein, IKAP (Rothwarf et al.,
1998
; Cohen et al.,
1998
) are also part of the complex. The critical step in the
signalling pathway is activation by phosphorylation of IKK
and
IKKß, which in turn phosphorylate the I
Bs at N-terminal serine
residues. Other cellular signals, such as that from p53 activation
(Ryan et al., 2000
), activate
NF-
B through IKK phosphorylation via a different cellular signalling
pathway involving Raf1, MEKK1 and p90RSK
(Ghoda et al., 1997
).
Phosphorylated I
B proteins are ubiquitinated by ß-TR-CP variants
(Spencer et al., 1999
) leading
to their degradation by the 26S proteasome
(Yaron et al., 1998
;
Coux and Goldberg, 1998
).
Following phosphorylation and degradation of the I
B proteins, the
NF-
B is released and its nuclear localisation sequence (NLS) becomes
unmasked, allowing the translocation of the NF-
B to the nucleus. It has
previously been suggested from studies in insect cells that the
NF-
B-I
B complex is the preferential substrate for
phosphorylation and degradation of the I
B by the IKKs, and that free
I
B alone is less efficiently phosphorylated
(Zandi et al., 1998
). This
would suggest that I
B bound to NF-
B would be preferentially
degraded, and that free I
B would be less efficiently degraded when the
IKKs are activated. In the nucleus, the NF-
B binds to a set of related
binding sites in the promoters of target genes. Each different NF-
B
complex has slightly different affinities for each specific DNA binding
sequence (reviewed by Zandi and Karin,
1999
). One of the genes that is activated by NF-
B is the
gene encoding I
B
. It is thought that newly synthesised
I
B
enters the nucleus and binds to NF-
B. The complex is
then relocated to the cytoplasm by CRM1-dependent nuclear export
(Arenzana-Seisdedos et al.,
1995
; Rodriquez et al.,
1999
).
Although a great deal has been learned about the individual parts of the
biochemistry of this signalling pathway, it is still unclear what factors
control the speed and longevity of the transcriptional response. To address
this, non-invasive tools are required to measure the different stages of the
signalling pathway in real-time in living cells. Recently, a number of studies
have used fluorescent proteins to report on the localisation and dynamics of
NF-B signalling. GFP-p105 was shown to translocate to the nucleus in
approximately 20 minutes after treatment with TNF
or hydrogen peroxide
(Tenjinbaru et al., 1999
). The
expression of a fusion protein between I
B
and EGFP
(Li et al., 1999
) also
demonstrated rapid degradation of the I
B
-EGFP signal in response
to NF-
B activating agents such as TNF
and the phorbol ester
polymyristate acetate. The use of dual fluorescent protein fusions between p65
and I
B
and different coloured GFP proteins allowed the analysis
of the biophysical basis of the interaction between these proteins using
fluorescent resonance energy transfer (FRET)
(Schmid et al., 2000
). To
investigate the kinetics of these processes in living cells, Carlotti and
colleagues first used a p65-EGFP fusion construct to show that in response to
IL-1ß stimulation, the kinetics of the response were sensitive to
p65-EGFP levels of expression. The levels of p65-EGFP were also critical for
the NF-
B-derived anti-apoptotic effect
(Carlotti et al., 1999
). In a
further study they used fluorescent fusion proteins with both p65 and
I
B
to confirm the previous suggestion that these proteins
undergo dynamic shuttling between the nucleus and cytoplasm, which is
associated with dissociation of the transcription factor and the inhibitor
within the cytoplasm (Carlotti et al.,
2000
).
In the present study, we have applied fluorescence imaging of p65 and
IB
together with luminescence imaging of NF-
B-dependent
transcription to study the real-time kinetics of the processes underlying
NF-
B-regulated transcription in living cells. We have used green
fluorescent protein and red fluorescent protein (dsRed)
(Matz et al., 1999
) chimeras
of I
B
and p65 together with firefly luciferase as a reporter
gene to investigate the timing of I
B
degradation, p65
translocation and NF-
B-dependent transcriptional activation in single
living cells. Studies of the kinetics of p65 translocation and
I
B
degradation in single or dual-transfected cells provided
functional kinetic data to support the hypothesis that degradation of
I
B
in the cytoplasm (or its targeting for degradation) occurs
preferentially when the I
B
is bound to NF-
B. Moreover, we
show that the timing of the translocation of p65 into the nucleus, in response
to TNF
stimulation, is critically dependent on the ratios of these
proteins. We show that LMB inhibition of nuclear export leads to rapid nuclear
accumulation of I
B
and slower nuclear accumulation of p65.
Treatment of cells with TNF
1 hour after LMB addition gives rise to
rapid and stable nuclear accumulation of p65 and normal transient
TNF
-activation-dependent kinetics of NF-
B-dependent
transcription. These results suggest that nuclear localisation of p65 is not
itself sufficient for stable NF-
B-dependent transcription and that a
further factor other than I
B concentration in the nucleus may be
required for the inhibition of NF-
B-dependent transcription.
![]() |
Materials and Methods |
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Plasmids
All plasmids were propagated using E. coli DH5 and purified
using Qiagen Maxiprep kits (Qiagen, UK). pNF-
B-Luc (Stratagene, UK)
contains five repeats of an NF-
B-sensitive enhancer element upstream of
the TATA box, controlling expression of luciferase. p65-EGFP contains a 1.6 kb
p65 cDNA cloned into the HindIII-BamHI site of pEGFP-N1
(kindly donated by M. Rowe, UWCM, Cardiff). This expresses a C-terminal
p65-EGFP fusion protein under the control of the human CMV immediate early
(hCMV-IE) promoter. pI
B
-EGFP (Clontech, UK) contains a fusion of
I
B
to EGFP under the control of the hCMV-IE promoter. p65-dsRed
was produced by inserting a 1.6 kb p65 HindIII-BamHI
fragment from p65-EGFP into the respective sites in the multiple cloning site
of pdsRed1-N1 (Clontech), producing an in-frame C-terminal fusion of p65 to
dsRed under the control of the hCMV-IE promoter.
Cell culture and transfection
HeLa Cells (ECACC No. 93021013) were grown in Minimal Essential Medium with
Earle's salts, plus 10% fetal calf serum, and 1% nonessential amino acids at
37°C, 5% CO2. For confocal microscopy and fluorescence
microscopy, cells were plated on 35 mm Mattek dishes (Mattek, USA) at
2.4x104 cells per plate in 2 ml medium. After 24 hours, cells
were transfected with appropriate plasmid(s) using Fugene 6 (Boehringer
Mannheim/Roche, Germany) following the manufacturer's recommendations. The
optimised ratio of DNA:Fugene 6 used for such transfections was 1 µg DNA
with 2 µl Fugene 6. This DNA concentration was maintained for single and
dual transfections with fluorescent protein expression vectors (i.e. 0.5 µg
of each plasmid). For triple transfections with two fluorescent protein
expression vectors and the NF-B-Luc reporter vector, 0.1 µg of each
fluorescent protein expression vector was used together with 0.8 µg of the
NF-
B-Luc expression vector DNA.
For microtitre plate-based luminescence assays of luciferase expression
from the pNF-B-Luc reporter plasmid, 1.4x104 cells
were seeded in 1 ml of medium into each well of a 24-well plate (Falcon,
Becton Dickinson, USA) and grown for 24 hours prior to transfection. Cells
were transfected for 24 hours using Fugene 6, at an optimised ratio of 0.5
µg pNF-
B-Luc to 1 µl Fugene 6 per well.
Fluorescence microscopy
Confocal microscopy was carried out on transfected cells in Mattek dishes
in a humidified CO2 incubator (at 37°C, 5% CO2)
using a Zeiss LSM510 with a 40x phase contrast oil immersion objective
(numerical aperture=1.3). Excitation of EGFP was performed using an Argon ion
laser at 488 nm. Emitted light was reflected through a 505-550 nm bandpass
filter from a 540 nm dichroic mirror. dsRed fluorescence was excited using a
green Helium Neon laser (543 nm) and detected through a 570 nm long-pass
filter. Data capture and extraction was carried out with LSM510 version 2.1
software (Zeiss, Germany). For p65-EGFP and p65-dsRed fusion proteins, mean
fluorescence intensities were calculated for each time point for both nuclei
and cytoplasm. Nuclear:cytoplasmic fluorescence intensity ratios were
determined relative to the initial ratio at t=0 minutes. For
IB
-EGFP fusion proteins, mean cellular fluorescence intensities
were calculated at each time point per cell, and fluorescence intensity
relative to starting fluorescence was determined for each cell.
Widefield fluorescence microscopy (Fig. 5A) was carried out on a Zeiss Axiovert S100 TV microscope under the same conditions as described for confocal microscopy, except that fluorescence excitation was carried out using a monochromator (Kinetic Imaging, UK) at 558 nm (±2 nm) for dsRed. Fluorescence emission was captured through a Texas Red filter block using a Hamamatsu C4742-98 CCD camera (Hamamatsu, Japan). Data acquisition and analysis were carried out with AQM2000 software (Kinetic Imaging). Analysis involved determination of mean fluorescence intensities for each nucleus and cytoplasm at each time point.
|
Microplate luminescence assays of living cells
For microtitre-plate-based living cell luciferase assays, 1 mM luciferin
was added to the medium 12 hours after addition of transfection reagent.
Luminescence was assayed a further 12 hours after luciferin addition to the
cells. Measurements of luminescence from 24-well plates were performed using a
Lumistar luminometer (BMG Labtechnologies, UK), using an integration time of
10 seconds per well per time point. In between successive time points the
microtitre plates were replaced in a tissue culture incubator at 37°C, 5%
CO2.
Combined fluorescence and luminescence microscopy
For triple-parameter imaging, the cells were transfected as described above
with 0.1 µg of each fluorescent protein expression vector and 0.8 µg of
NF-B-Luc reporter vector. Twenty-four hours later, firefly luciferin
(Biosynth, Switzerland) was added to the medium to a final concentration of
0.5 mM. Cells were used after a minimum luciferin incubation of 4 hours.
Confocal microscopy was performed as described above for LMB treatment and 40
minutes of TNF
incubation. The confocal microscope was then switched
off to allow luminescence imaging.
Luminescence imaging was carried out using a Hamamatsu 4880-65 liquid nitrogen cooled CCD camera attached to the top port of the confocal microscope. AQM2000 software was used for image acquisition and analysis. Images were acquired using 30 minute integration times. Successive luminescence images and control blank images were used to automatically remove dark noise and noise from random cosmic events. For dual confocal and luminescence microscopy, the same cells were identified from bright field images.
Treatment of cells with TNF, inhibitors and leptomycin B
Treatment of cells with TNF (10 ng/ml final concentration) was
carried out immediately before microscopy by replacing one tenth of the medium
volume in the dish with the appropriate solution. Inhibitors were added prior
to TNF
treatment in the same manner. Each experiment was carried out at
least twice with at least four cells obtained per replicate. Leptomycin B
(Sigma, UK) was dissolved in methanol and added to a final concentration of 10
ng/ml (18 nM).
![]() |
Results |
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|
To confirm the functional significance of these observations, the effects
of the Bay11-7082 and SN50 inhibitors on translocation of the p65-EGFP fusion
protein were investigated using concentrations previously shown to
significantly inhibit TNF-induced expression from an NF-
B-Luc
expression vector (data not shown). The cell-permeable peptide SN50 contains
the sequence of the NLS of NF-
B and thus competitively inhibits
NF-
B nuclear translocation (Lin et
al., 1995
). Bay11-7082 irreversibly inhibits TNF
-induced
phosphorylation of I
B
(Pierce et al., 1997
), thereby
preventing the subsequent ubiquitin-mediated I
B
degradation. No
visible effects of inhibitor addition prior to stimulation with TNF
were observed (data not shown). Preincubation of cells with 18 µM SN50 for
15 minutes prior to stimulation with TNF
gave rise to partial
inhibition (approximately 50%) of nuclear translocation of p65-EGFP
(Fig. 1), indicating that
import of the fusion protein p65-EGFP is effected by the NLS-mediated import
pathway. The related control peptide, SN50M, which has two amino acid
substitutions in the NLS relative to SN50, showed a slight inhibitory affect
on p65-EGFP translocation (Fig.
1B). An ANOVA analysis of these data showed that after 40 minutes
of TNF
treatment, both the control and SN50M treated cells were
significantly different from SN50, but not from one another
(P=0.006). Cells treated with 12.5 µM Bay11-7082 30 minutes prior
to stimulation with TNF
also showed very significant inhibition of
p65-EGFP translocation, indicating that p65-EGFP is bound to I
B
and held in the cytoplasm, only to be released upon phosphorylation of
I
B
. Thus, the fusion protein p65-EGFP appears to possess the
functional characteristics of an NF-
B protein.
To observe the kinetics of IB degradation following TNF
treatment, HeLa cells were transiently transfected with an
I
B
-EGFP fusion protein. Cells expressing the
I
B
-EGFP fusion protein showed mainly cytoplasmic fluorescence 24
hours post transfection (Fig.
2A). A rapid decay in fluorescence was observed upon stimulation
with TNF
. The half-time of degradation of this protein was determined
by quantitative fluorescence analysis to be 26.7±2.8 minutes
(n=14; Fig. 2B). The
degradation of the I
B
-EGFP fusion protein following TNF
stimulation was inhibited by treatment with 12.5 µM Bay 11-7082
(Fig. 2A,B), but not with 18
µM SN50 (Fig. 2B). This
observation confirms that the inhibitory action of SN50 on the translocation
of p65 (Fig. 1) is downstream
of I
B
phosphorylation. The inhibitory effect of Bay11-7082 (the
inhibitor of I
B
phosphorylation), but not SN50 (the inhibitor of
p65 translocation), supports the hypothesis that the observed fluorescence
decrease is due to TNF
-induced degradation of the I
B
-EGFP
fusion protein via phosphorylation of I
B
.
|
Increased expression of p65-dsRed enhances the rate of degradation of
IB
-EGFP following TNF
treatment
We investigated whether extended kinetics of IB
degradation
following I
B
-EGFP overexpression could be modulated by increased
levels of p65 expression. To test this hypothesis, we constructed a fusion
protein between the red fluorescent protein, dsRed and p65. This allowed
independent measurement of p65-dsRed translocation and I
B
-EGFP
degradation as well as quantitative measurement of the starting levels of
expression of both fusion proteins in each cell. Addition of TNF
to the
cells produced the expected overall response from both fusion proteins,
indicating that the two processes could be independently measured in the same
cell (Figs 3,
4). In dual transfected cells
expressing I
B
-EGFP and p65-dsRed, I
B
-EGFP
degradation reached 50% of the initial cellular fluorescence in
13.5±1.7 minutes (n=9; Fig.
3A). This was significantly shorter than the t
for I
B
-EGFP degradation in single transfected cells (see above)
and in dual transfected cells expressing I
B
-EGFP and control
dsRed protein (t
=18.2±2.2 minutes, n=12;
Fig. 3A). The rate of
I
B
-EGFP degradation in dual transfectants with p65-dsRed
therefore more closely resembled the rate of native I
B
degradation in untransfected cells (Sun et
al., 1993
). The observation that the rate of I
B
degradation is accelerated in the presence of p65-dsRed fusion protein
provides the first functional evidence from mammalian cells to support the
hypothesis originally proposed following work in insect cells
(Zandi et al., 1998
) that IKK
can efficiently phosphorylate only NF-
B-I
B dimers, as opposed to
unbound I
B.
|
|
Increased levels of IB
-EGFP delay nuclear translocation
of p65-dsRed following TNF
treatment
We next investigated whether the level of IB
fusion protein
could modulate the rate of nuclear translocation of p65-dsRed. The rate of
p65-dsRed translocation was indistinguishable from that seen with p65-EGFP
translocation (data not shown). Cells expressing p65-dsRed with a control EGFP
expression vector gave rise to significantly more rapid p65-dsRed
translocation than that observed in cells transfected with p65-dsRed together
with I
B
-EGFP (Fig.
3B). The half-time for translocation of p65-dsRed in cells
co-transfected with I
B
-EGFP was 24±2.9 minutes, as
opposed to a much shorter half time of 11.6±1.1 minutes for dual
transfectants expressing p65-dsRed with pEGFP-N1. This suggests that the ratio
of p65 and I
B
in cells determines the kinetics of p65-EGFP
nuclear accumulation.
To provide further evidence to support this conclusion, we studied the rate
of p65 translocation in dual-transfected cells expressing markedly different
levels of IB
-EGFP. Cells expressing significantly higher levels
of I
B
-EGFP showed slower I
B
-EGFP degradation and
delayed p65-dsRed translocation compared with cells expressing much lower
levels of I
B
-EGFP. This is illustrated in
Fig. 4, which shows two pairs
of cells (marked a and b) with approximately threefold
variation in their levels of I
B
-EGFP, but similar expression
levels of p65-dsRed (as determined by fluorescence quantification). Cell pair
a had lower levels of I
B
-EGFP relative to cell pair
b and showed faster translocation of p65 following TNF
stimulation compared with cells b, which had higher levels of
I
B
. A different panel of cells are shown in the movie file (see
http://jcs.biologists.org/supplemental
), which shows a group of cells with widely varying initial
p65-dsRed:I
B
-EGFP ratios. The rate of p65-dsRed nuclear
accumulation is again seen to be inversely proportional to the level of
I
B
-EGFP in each cell. These data suggest that the ratio of
I
B
and p65 plays a role in determining the kinetics of the
NF-
B transcriptional response to TNF
.
Monitoring of long-term p65 dynamics coupled with transcriptional
activation
To investigate the longer term kinetics of activation of the NF-B
signalling pathway by TNF
, we studied the kinetics of p65 localisation
over periods of several hours following TNF
treatment
(Fig. 5A). Following initial
nuclear translocation of p65-dsRed, the vast majority of the transcription
factor was observed to move back into the cytoplasm within 5 hours. To
determine whether this was caused by genuine protein export or simply
degradation of the nuclear protein followed by de novo synthesis of new
cytoplasmic p65, we compared the kinetics of export using p65-dsRed and
p65-EGFP (data not shown). A major difference between the properties of these
two fluorescent proteins lies in the time over which the fluorescent protein
acquires its fluorescence. DsRed takes several hours longer than EGFP for its
chromophore formation (Baird et al.,
2000
). The fact that the kinetics of apparent nuclear export of
the p65-fusion protein were indistinguishable when using either fluorescent
protein (data not shown) strongly suggests that this corresponds to the export
of pre-synthesised nuclear protein rather than new fluorescent protein
synthesis. These data suggest that the p65 protein is present in the nucleus
for only a relatively short period of time and that the change in timing of
p65 translocation due to I
B
levels (up to 12 minutes) results in
a delay in transcription upregulation that is of a significant duration in
comparison to the length of time that p65 resides in the nucleus.
To investigate the relationship between this transient p65 occupation of
the nucleus and the timing of transcription, we investigated the timing of
transcription in living cells. Luminescence assays of cells plated in a
24-well plate and treated with luciferin indicated that the timing of
transcription was transient with a peak at around 4-5 hours after TNF
addition (Fig. 5B). Allowing
for the typical 4 hour delay in synthesis of the luciferase fusion protein
(data not shown), these observations suggested that the timing of
transcription correlated closely with the occupancy of the nucleus by
NF-
B.
Measurement of p65 translocation, IB
degradation and
NF-
B-directed transcription using combined confocal microscopy and
luminescence imaging
To investigate the relationship between signalling and transcription in
single cells we used a low light level camera attached to a confocal
microscope. HeLa cells were transfected with p65-dsRed, IB
-EGFP
and an NF-
B-Luc expression vector. The cells were initially monitored
for fluorescence after treatment with TNF
and then the resulting
luminescence over the following 10 hours was measured in the same cells by
luminescence imaging. Analysis of single cells indicated that similar dynamics
of translocation of p65 and degradation of I
B
(Fig. 6A,B) were seen compared
with those described above. In agreement with the results obtained from cell
population analysis (Fig. 5B),
the individual cells gave rise to a consistent transient luminescence response
indicating rapid activation and repression of NF-
B-directed
transcription (Fig. 6B,C). The
analysis of the timing of induction in cells with widely varying levels of
luminescence intensity (Fig. 6,
cells ac) suggested that this timing was maintained irrespective of the level
of transfection with the luciferase reporter plasmid.
|
Rates of nuclear accumulation of p65 and IB
following
inhibition of nuclear export
To investigate the relationship between transcription and nuclear export of
p65 we used the CRM1-dependent inhibitor of nuclear export, LMB. Treatment of
cells with this inhibitor led to nuclear accumulation of both p65-dsRed and
IB
-EGFP (Rodriquez et al.,
1999
; Carlotti et al.,
2000
), supporting the hypothesis that these proteins are involved
in nucleo-cytoplasmic shuttling even in unstimulated cells
(Johnson et al., 1999
;
Huang et al., 2000
). Analysis
of the rate of nuclear accumulation of these proteins showed significantly
more rapid accumulation of I
B
than p65-dsRed
(Fig. 7A) in agreement with
previous results (Carlotti et al.,
2000
). To show that LMB treatment led to stable localisation of
p65 and I
B
in the nucleus, cells were transfected with p65-dsRed
and I
B
-EGFP and treated with LMB. Both p65-dsRed and
I
B
-EGFP were maintained in the nucleus for more than 8 hours
(Fig. 7B, top). When cells were
treated with 10 ng/ml TNF
1 hour after LMB treatment, there was a more
rapid translocation of the p65-dsRed into the nucleus followed by its
maintenance in the nucleus for 8 hours
(Fig. 7B, bottom).
|
Analysis of protein dynamics and transcription in leptomycin
B-treated cells
After long periods of LMB treatment (6 hours or more) when both
IB
-EGFP and p65-dsRed were localised to the nucleus, TNF
treatment did not give rise to nuclear degradation of I
B
-EGFP or
NF-
B-dependent transcription (data not shown). This suggested that some
p65 must remain in the cytoplasm in order for a response to TNF
to
occur. (NF-kB-independent transcription from an exogenous promoter was not
inhibited, suggesting that this observation was not caused by non-specific
transcriptional inhibition following long-term LMB treatment.) One hour after
LMB treatment, there was significant nuclear I
B
-EGFP
accumulation, but a large proportion of the p65-dsRed remained present in the
cytoplasm (Figs 7,
8). When these cells were
stimulated with TNF
, there was nuclear translocation of the remaining
cytoplasmic pool of p65-dsRed, indicative of a normal response. Owing to the
continued presence of the LMB, the p65-dsRed then remained in the nucleus,
since nuclear export was inhibited (Figs
7,
8). (Note that the scale in
Fig. 8B is nuclear/cytoplasmic
fluorescence, rather than simply nuclear fluorescence as in
Fig. 7A, making relative
movement of p65 appear less significant in the first hour after LMB
treatment.) There was degradation of I
B
-EGFP in both the
cytoplasm and nucleus, although 40 minutes after TNF
treatment, some
nuclear I
B
-EGFP fluorescence remained, whereas cytoplasmic
I
B
-EGFP fluorescence was undetectable
(Fig. 7B;
Fig. 8A,B). These cells also
displayed a normal transient stimulation of NF-
B-dependent
promoter-directed transcription in response to TNF
(Fig. 8B). Cells transfected
with a control promoter and treated with LMB under similar conditions
indicated that the transient time course of luciferase expression was not due
to non-specific effects of LMB treatment (data not shown).
|
![]() |
Discussion |
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We showed that both the p65-EGFP and p65-dsRed fluorescent fusion proteins
gave rise to the nuclear translocation in response to TNF treatment,
which is characteristic of the functional endogenous protein. Ding et al.
previously reported an endogenous p65 nuclear translocation half time of 7-8
minutes in HeLa cells following TNF
stimulation
(Ding et al., 1998
). In
comparison, we obtained a longer half time of 19±2.9 minutes for
nuclear translocation of p65-EGFP in singly transfected cells
(Fig. 1A) in agreement with
other studies using a p65-EGFP fluorescent fusion protein and stimulation with
IL-1ß (Carlotti et al.,
1999
; Carlotti et al.,
2000
). One explanation is that high expression of the p65 fusion
protein results in these differences. When we studied the translocation of
p65-dsRed (or p65-EGFP) in cells co-transfected with a control fluorescent
protein expression vector (Fig.
3B), the half-time of translocation was faster (11.6±1.3
minutes). In the dual transfected, compared with the single transfected cells,
expression of the p65-fluorescent protein was markedly reduced (typically 70%
of the single transfectant levels), perhaps due to promoter or plasmid
competition. The difference between our observed translocation times and those
of Ding et al. may therefore represent the effect of the level of p65
expression on the dynamics of its translocation.
The IB
-EGFP fusion protein is subject to TNF
-induced
reduction of fluorescence, which can be related to the degradation of the
endogenous I
B
protein. Expression of the I
B
-EGFP
fusion protein for extended periods in HeLa cells was found to induce
apoptosis (data not shown). This I
B
-specific effect supports the
hypothesis that the I
B
moiety is functional as a part of the
fusion protein. The relationship between the relative levels of the p65- and
I
B
-fluorescent proteins and the related changes in the kinetics
of the response of both proteins to TNF
treatment suggest that these
proteins have retained the ability to interact with each other (see below).
The observation of expected inhibitory effects using the NF-
B
inhibitors SN50 and Bay11-7082 further supported the hypothesis that the
fluorescent fusion proteins retained endogenous protein function.
The half-time of fluorescence degradation in cells transfected with
IB
-EGFP alone (26.7±2.8 minutes;
Fig. 2B) and cells
co-transfected with a control dsRed expression vector (18.2±2.2
minutes; Fig. 3A) was
significantly slower than that described in previous studies
(Li et al., 1999
;
Henkel et al., 1993
;
Sun et al., 1993
). Western
blot analysis of cells transfected with I
B
-EGFP showed that this
delay was not due to cleavage of EGFP from the fusion protein, as no bands
smaller than the fusion protein were observed when probing with an anti-EGFP
antibody, and endogenous I
B
degradation occurred at the same
rate as I
B
-EGFP degradation in transfected cells (data not
shown). A previous study observed a 5 minute half life of an
I
B
-EGFP fusion protein after treatment with 100 ng/ml TNF
(Li et al., 1999
). Expression
of I
B
-EGFP in that study was under inducible control, which may
have prevented accumulation of high levels of I
B
-EGFP in the
cell. Single transfectants with I
B
-EGFP in our study showed
approximately 40% higher expression of fluorescent I
B
-EGFP than
cells cotransfected with a control dsRed expression vector. The lower
expressing dual-transfected cells showed faster degradation of
I
B
-EGFP than the higher-expressing single transfectants. Higher
levels of I
B
expression may therefore saturate the I
B
phosphorylation, ubiquitination or degradation pathways.
We observed that co-expression of p65-dsRed together with exogenous
IB
-EGFP gave rise to significantly faster degradation of
I
B
-EGFP fluorescence (half life 13.5±1.7 minutes;
Fig. 3A) compared with single
(26.7±2.8 minutes; Fig.
2B) or control dual transfections (18.2±2.2 minutes,
P=0.024; Fig. 3A).
Therefore, these data suggest that higher levels of p65 expression
specifically and significantly increase the rate of I
B
degradation. This suggests that NF-
B-I
B complexes may be the
natural substrates for an IKK rather then I
B proteins alone,
confirming, in living mammalian cells, previous results obtained from
phosphorylation studies in insect cells
(Zandi et al., 1998
).
We also show that higher levels of IB
-EGFP significantly
delay the timing of p65-dsRed nuclear translocation
(Fig. 3B;
Fig. 4). For
p65-dsRed+I
B
-EGFP dual transfections the half time of
translocation was 24.5±2.9 minutes compared with 11.6±1.3
minutes in p65-dsRed + control EGFP dual transfections (P=0.012).
These data therefore suggest that high levels of I
B
specifically
delay nuclear import of p65.
The effect of the ratio of two proteins on the timing of nuclear signalling
is a potentially important mechanism by which cells may respond differentially
to the same signal. This may have important functional consequences in cells
treated with TNF, since TNF
is known to elicit a death response
leading to apoptosis through the TNFR. However, the NF-
B response to
TNF
is known to protect cells from apoptosis
(Foo and Nolan, 1999
). It is
therefore possible that a delayed NF-
B response in cells with a high
I
B:NF-
B ratio might be more likely to lead to apoptosis. This
possibility remains to be investigated. We also show that the timing of p65
nuclear occupancy is approximately 2.5 hours as measured by half maximum to
minimum translocation levels, or 10 minutes (or less) as measured by peak
nuclear p65-dsRed fluorescence (Fig.
5A). The observed delay in p65 import in cells expressing high
I
B:p65 ratios might therefore contribute to the overall timing of the
NF-
B signal and its functional significance.
To investigate the relationship between p65 translocation, IB
translocation/degradation and transcription in the same cells, we applied a
novel technique involving two-colour fluorescence and luminescence imaging.
The time-course of transcription from the NF-
B consensus promoter was
found to be transient, as previously observed by cell population analysis
(Arenzana-Seisdedos et al.,
1995
). The repression phase of the transient NF-
B
transcription response has been suggested to involve the induction of
endogenous synthesis of I
B
following NF-
B activation
(Place et al., 2001
). This may
result in nuclear accumulation of the newly synthesised I
B
,
which binds to NF-
B, inhibiting transcription and leading to
CRM-1-dependent nuclear export of the inactive I
B
-NF-
B
complex into the cytoplasm. To investigate the relationship between the timing
of protein movement and transcription, we treated the cells with the inhibitor
of CRM-1-dependent nuclear export, LMB. This led to import of p65-dsRed and
I
B
-EGFP into the nucleus of the cells, caused by inhibition of
the export component of normal nucleo-cytoplasmic shuttling. As reported
previously (Carlotti et al.,
2000
), the rate of import of I
B
was significantly
higher than that of the p65 fluorescent fusion protein, suggesting that these
proteins enter the nucleus by separate pathways and as separate entities.
Treatment with LMB did not activate basal NF-
B-dependent transcription.
After a 6 hour treatment with LMB, the NF-
B-dependent transcription
response to TNF
was blocked, but not general transcription from a
control promoter (data not shown). However, cells that had been treated for
only 1 hour with LMB before treatment with TNF
still showed cytoplasmic
p65 and were able to elicit NF-
B-dependent transcription. Under these
conditions, we observed I
B
-EGFP degradation both in the
cytoplasm and to a lesser extent in the nucleus, as well as rapid and stable
nuclear accumulation of the remaining cytoplasmic p65-dsRed. The demonstration
of I
B
degradation in the nucleus may support the recent
suggestion that this is an important component of NF-
B regulation
(Renard et al., 2000
),
although we do not see significant I
B
degradation (or
transcriptional activation) in response to TNF
at later times following
LMB treatment. Despite the accumulation of p65 in the nucleus (since nuclear
export was inhibited with LMB), the transcription response to TNF
stimulation was transient, with similar kinetics to those observed in cells
that had not been treated with LMB. This confirms that the dynamics of
transcription are not dependent simply on the nuclear localisation of p65.
Since a transient transcription response still occurs in the presence of a
high concentration of remaining nuclear IB
after 1 hour of LMB
treatment, it seems likely that a further parameter may affect the rate of
I
B
degradation in the nucleus and the ability of nuclear
I
B
to regulate transcription. One possibility is that functional
transcription requires a further cytoplasmic event to occur, such as
phosphorylation of the p65 subunit of NF-
B. The functional importance
of phosphorylation of serine residues on p65 has been demonstrated in a number
of studies (Wang and Baldwin,
1998
; Zhong et al.,
1998
; Anrather et al.,
1999
; Mercurio et al.,
1997
; Sakurai et al.,
1999
; Fognani et al.,
2000
; Martin and Fresno,
2000
; Wang et al.,
2000
; Jang et al.,
2001
; for a review, see
Schmitz et al., 2001
). This
might explain the observation in the present experiments that transcriptional
upregulation from the NF-
B promoter is only seen at times when there is
still significant p65 remaining in the cytoplasm following LMB treatment.
Recently, cells from knockout mice that lack the gene encoding glycogen
synthase kinase-3ß protein were shown to elicit translocation of p65-p50
heterodimers to the nucleus, but did not give an NF-
B transcriptional
response (Hoeflich et al.,
2000
). Cytoplasmic modifications of NF-
B proteins may
therefore modulate transcriptional activity in conjunction with NF-
B
nuclear translocation.
The present results suggest that treatment with LMB does not prevent the
post-induction repression of NF-B-dependent transcription as suggested
previously (Rodriquez et al.,
1999
). Rather, the kinetics of the transient reporter gene
response that we observe is remarkably similar in 1 hour LMB pre-treated cells
and non-LMB-treated cells. This suggests that newly synthesised
I
B
may enter the nucleus and rapidly repress transcription,
despite the inhibition of nuclear export resulting in long-term nuclear
localisation of p65. It might be expected that we should see significant
inhibition of the initial phase of transcription induction by the accumulated
excess of nuclear I
B
that is present at the time of TNF
induction. However, this does not seem to occur. The level of nuclear
I
B
-EGFP remained significant (albeit lower due to some nuclear
degradation) 40 minutes after TNF
stimulation. These results suggest
that a further factor that might include p65 or I
B
interactions
with other proteins [such as hnRNPA1 (Hay
et al., 2001
)], co-activators or co-repressors, such as silencing
mediator of retinoic acid and thyroid hormone receptor [SMRT
(Jang et al., 2001
;
Jong and Privalsky, 2000
)], or
phosphorylation of p65 (Jang et al.,
2001
) may further regulate the timing of these processes.
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Acknowledgments |
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Footnotes |
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References |
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