1 Biosciences Building, School of Biological Sciences, University of Liverpool,
Crown Street, Liverpool L69 7ZB, UK
2 Department of Medicine, University of Manchester, Stopford Building,
Manchester M13 9PT, UK
3 Lead Generation, Molecular Biology, AstraZeneca R&D Charnwood, Bakewell
Road, Loughborough LE11 5RH, UK
* Author for correspondence (e-mail: mwhite{at}liv.ac.uk)
Accepted 6 March 2003
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Summary |
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Key words: NF-B, STAT6, Glucocorticoid receptor, Signal transduction, Fluorescent protein fusions, Confocal microscopy
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Introduction |
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Downregulation of NF-B-mediated transcription is thought to occur
through newly synthesized I
B entering the nucleus and binding
NF-
B. The NF-
B/I
B complex is then translocated to the
cytoplasm by CRM1-dependent nuclear export
(Arenzana-Seisdedos et al.,
1995
; Rodriquez et al.,
1999
). Downregulation does not require cytoplasmic relocalization
of NF-
B, because transcription can be switched off even when
NF-
B is trapped in the nucleus after stimulation
(Nelson et al., 2002a
).
The glucocorticoid receptor (GR) is activated by binding glucocorticoid
(GC) at its C-terminal ligand-binding domain
(Hollenberg and Evans, 1988).
Activated GR can then dimerize and translocate to the nucleus and recruit
co-activator proteins such as GRIP1 and SRC1, to bind GC response elements
(GRE) and to activate transcription (Hong
et al., 1997
; McKenna et al.,
1999
). GR localization within a cell appears to be dynamic, with
nucleocytoplasmic shuttling of both ligand-bound GR and `resting' GR in
hormone-free cells (Madan and DeFranco,
1993
; Defranco et al.,
1995
; Htun et al.,
1996
). Activated GR rapidly moves to the nucleus because of the
presence of two NLSs (Picard and Yamamoto,
1987
) using the cytoskeleton and at least one chaperone, heat
shock protein 90 (Yang and DeFranco,
1996
; Galigniana et al.,
1998
).
GCs are potent anti-inflammatory agents. The GR binds to other
transcription factors, including NF-B p65
(Ray and Prefontaine, 1994
;
Caldenhoven et al., 1995
) and
prevents transactivation of their target genes, but it has been suggested that
this does not alter the occupancy of the DNA response elements
(Nissen and Yamamoto, 2000
).
It has been possible to dissociate repression of NF-
B from
transactivation through targeted mutation of GR, indicating a requirement for
distinct regions of the protein for these functions
(Ray et al., 1999
;
Heck et al., 1994
;
Heck et al., 1997
;
Bledsoe et al., 2002
).
Interleukin 4 (IL-4) exerts multiple effects on the immune system via
activation of signal transducer and activator of transcription 6 (STAT6),
which is involved in immunoglobulin class switching and T-helper cell type-2
differentiation. STAT6 and NF-B p50 have been shown to bind one
another, but it is not known whether this interaction occurs in the cytoplasm
or the nucleus (Shen and Stavnezer,
1998
). Synergistic effects between the pathways have been
demonstrated in the Ig germline
promoter
(Delphin and Stavnezer, 1995
).
There have been no reports describing an interaction between STAT6 and
NF-
B p65, which is the subunit of the p65/p50 heterodimer that contains
a transactivating domain (Schmitz and
Baeuerle, 1991
).
Conversely, IL-4 has been shown to suppress tumour necrosis factor
(TNF
)-induced expression of the E-selectin gene through STAT6 competing
for an overlapping consensus sequence within a dual NF-
B enhancer
element (Bennett et al., 1997
).
In addition, IL-4 has been shown to inhibit TNF
-induced transcription
from the interferon response factor 1 (IRF-1) gene promoter
(Ohmori and Hamilton, 2000
) by
competition of NF-
B for the co-activator CBP (CREB-binding protein). In
cells transfected with a mutant of STAT6 in which its transactivation domain
had been deleted, IL-4 was unable to inhibit TNF
-stimulated
transcription. More recently, IL-4 has been shown to inhibit
osteoclastogenesis through STAT6-dependent inhibition of RANKL-dependent
activation of NF-
B (Abu-Amer,
2001
; Wei et al.,
2002
). These studies have shown that STAT6 inhibits I
B
degradation, so preventing NF-
B moving to the nucleus. However, this
effect on I
B has only been described when IL-4 was applied for between
24 hours and 3 days. This suggested that STAT6 was probably inducing other
factors that feed back onto the NF-
B signalling pathway rather than
directly binding NF-
B components.
We have previously shown, using fluorescent fusion proteins, that the rate
of IB
degradation depends on p65 expression levels and that the
kinetics of p65 translocation are altered by I
B
overexpression
(Nelson et al., 2002a
). Direct
interaction between these proteins has been demonstrated through fluorescence
resonance energy transfer (FRET) (Schmid
et al., 2000
). These studies have confirmed the normal function of
p65 and I
B
fusion proteins and their usefulness for
investigating the principles of the pathway dynamics.
From the literature summarized above, it is clear that STAT6 and GR
interact functionally with NF-B. However, it is still not clear how,
where and when these interactions occur. We have used fluorescent protein
fusions to components of the three pathways to investigate the interactions
between GR and STAT6 with both p65 and I
B
proteins of the
NF-
B pathway. We have shown that activating either the GC or the IL-4
pathway resulted in decreased NF-
B-mediated transcription and inhibited
the nuclear localization of p65. However, this inhibition appeared to occur
through different mechanisms. GR increased the rate of p65 nuclear export
after stimulation with TNF
and had no effect upon I
B
,
whereas STAT6 appeared to inhibit p65 import after stimulation with
TNF
, which appeared to be due to direct inhibition of I
B
phosphorylation and degradation. Both methods of inhibition appear to operate
before the formation of transcription initiation complexes and occurred too
quickly for their response to be explained by transcriptional regulation.
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Materials and Methods |
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Plasmids
All plasmids were propagated using Escherichia 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 the expression of luciferase.
pTAT3-luc controls the expression of luciferase from a minimal alcohol
dehydrogenase promoter, with three copies of the glucocorticoid response
element from the tyrosine aminotransferase gene promoter. All fluorescent
protein expression plasmids are under the control of the human cytomegalovirus
immediate early promoter. pEGFP-N1 expresses enhanced green fluorescent
protein (EGFP) without any fusion protein attached (Clontech, UK). 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). p65-dsRed was
constructed as described previously
(Nelson et al., 2002a
). These
constructs express a C-terminal p65-fluorescent protein fusion.
pI
B
-EGFP (Clontech) contains a fusion of I
B
to
EGFP. pEGFP-GR expresses an N-terminal fusion of GR to EGFP
(Galigniana et al., 1998
).
pEGFP-STAT6 expresses an N-terminal fusion of human STAT6 to EGFP
(Nelson et al., 2002b
).
Cell culture and transfection:
HeLa cells (ECACC No. 93021013) were grown in minimal essential medium with
Earle's salts, plus 10% FCS and 1% nonessential amino acids at 37°C, 5%
CO2. For confocal microscopy, cells were plated on 35 mm Mattek
dishes (Mattek, USA) at 2x=104 cells per plate in 2 ml
medium. After 24 hours, cells were transfected with appropriate plasmid(s)
using Fugene 6 (Boehringer Mannheim/Roche) following the manufacturer's
recommendations. The optimized 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 microtitre-plate-based luminescence assays of luciferase expression
from the pNF-B-luc reporter plasmid, 1 x 104 cells were
seeded in 1 ml of medium into each well of 24 well plates (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 0.25 µg
DNA to 0.5 µl Fugene 6 per well.
Fluorescence microscopy
Confocal microscopy was carried out on transfected cells in Mattek dishes
in a Zeiss XL humidified CO2 incubator (37°C, 5%
CO2) using a Zeiss LSM510 Axiovert 200 microscope 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 band-pass filter from a 540 nm dichroic mirror.
dsRed fluorescence was excited using a green heliumneon laser (543 nm) and
detected through a 560 nm long-pass filter. Data capture and extraction was
carried out with LSM510 version 3 software (Zeiss, Germany). Treatment of
cells with TNF (10 ng ml-1 final concentration) was carried
out immediately prior to microscopy by replacing one tenth of the medium
volume in the dish with the appropriate solution. Pre-treatment with
dexamethasone, RU486 or IL-4 was carried out in the same manner. Each
experiment was carried out at least twice with at least four cells obtained
per replicate. For 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 0 minutes. For I
B
-EGFP fusion proteins,
mean cellular fluorescence intensities were calculated at each time point per
cell and the fluorescence intensity relative to starting fluorescence was
determined for each cell.
Reporter luminescence assays
Cells were plated in 24-well microtitre plates as described above. Wells
were treated for 6 hours (for NF-B-luc assays) or 18 hours (for
TAT3-luc assays) prior to harvesting the cells in 250 µl lysis buffer
(White et al., 1990
). Each
well was assayed in duplicate by transferring 100 µl of lysate into white
96-well plates (Greiner, UK), with ATP added to a final concentration of 1.25
mM. Luminescence was measured using a BMG Lumistar plate reader fitted with an
injector (BMG Labtechnologies, UK), which was used to add 100 µl of 1 mM
luciferin (Biosynth, Switzerland) to each well. Experiments were performed in
triplicate.
Western blotting
HeLa cells were seeded in 90 mm Petri dishes (Falcon, Becton Dickinson) at
1x106 per dish. 24 hours later, all plates were pre-treated
for 40 minutes with either 10 ng ml-1 IL-4 or medium prior to
treatment with TNF (10 ng ml-1). Following treatment, cells
were harvested in 750 µl lysis buffer (40 mM Tris-Cl, pH 6.8, 1% w/v SDS,
1% v/v glycerol, 1% v/v ß-mercaptoethanol, 0.01% w/v bromophenol blue) at
various times and the lysates boiled for 5 minutes. 50 µl of each lysate
was run on a 10% SDS-PAGE gel followed by blotting using a Biorad Protean II
electrophoresis and blotting apparatus (Biorad, UK), following the
manufacturer's instructions. Even loading of the gel was ascertained by
Coomassie staining. Detection of serine-32-phosphorylated I
B
with anti-phospho-I
B
antibody was performed following the
manufacturer's instructions.
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Results |
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Additionally, we wished to discover whether GFP-GR could also act as a
functional transcriptional repressor of the NF-B signalling pathway
(Fig. 1B). Treatment of
EGFP-expressing cells with dexamethasone was shown to inhibit the level of
TNF
-induced luciferase expression from pNF-
B-luc significantly
(95.2±1.7%). In cells expressing GFP-GR, the level of inhibition of
pNF-
B-luc expression by dexamethasone was also very significant
(98.1±1.04%) and could not be statistically distinguished from that
obtained with the control EGFP-expressing cells. The expression of EGFP-GR
together with the p65-EGFP significantly increased dexamethasone inhibition of
pNF-
B-luc expression (54.0±3.3%) relative to that obtained with
p65-EGFP alone (25.94±8.2%). This showed that expression of the EGFP-GR
fusion protein could enhance GC-mediated inhibition of NF-
B-mediated
signalling by TNF
.
Inhibition of the NF-B pathway by activated GR
In order to investigate the mechanism of GR inhibition of
NF-B-mediated transcription, we used noninvasive single-cell imaging to
monitor the localization of a p65-dsRed fusion protein
(Nelson et al., 2002a
).
Stimulation of HeLa cells expressing EGFP and p65-dsRed with TNF
showed
a phase of nuclear accumulation of p65 lasting
40 minutes. Subsequently,
the p65-dsRed fluorescent protein was exported back out of the nucleus to the
cytoplasm, reflecting a second phase of net nuclear export, which was almost
complete after
80 minutes (Fig.
2B, Fig. 3A). This
resulted in a period of nuclear occupancy (defined as half-maximal nuclear
import to half-maximal nuclear export) of 40.0±0.6 minutes
(Fig. 3C). Pretreatment of
EGFP-and p65-dsRed-expressing cells with 10 nM dexamethasone for 40 minutes
before TNF
stimulation resulted in a marked alteration in the time
course of p65-dsRed nuclear occupation
(Fig. 3A). This appeared to be
caused by earlier net nuclear export rather than an inhibition of nuclear
import, leading to a significantly shorter period of nuclear occupancy
(26.4±0.6 minutes, P<0.05;
Fig. 3C). Overexpression of
EGFP-GR (instead of the control EGFP) also resulted in a similarly decreased
period of TNF
-induced p65-dsRed nuclear occupation in cells pretreated
with dexamethasone (24.4±1.1 minute for cells pretreated with 10 nM
dexamethasone compared to 30.3±0.3 minutes for control pretreated
cells, P<0.05; Fig.
2A, Fig. 3B,C). The
expression of EGFP-GR also changed the dynamics of p65-dsRed nuclear occupancy
in response to TNF
in cells not pretreated with dexamethasone but this
reduction was less than in dexamethasone pretreated cells
(Fig. 3B,C). These results
suggested that activated GR was directly involved in the mechanism of
dexamethasone-stimulated export of p65.
|
|
Inhibition of NF-B signalling by the GR agonist RU486
We wished to determine whether this increased export of p65 required
GR-dimerisation, binding to DNA and activation of gene expression. The GR type
II agonist RU486 gives rise to nuclear import of GR without stimulating these
subsequent events (Beck et al.,
1993). Therefore, we pre-treated cells expressing EGFP-GR and
p65-dsRed with this agonist in place of dexamethasone. As shown in Figs
3B and 3C, this also led to a
decrease in the time of nuclear occupation of p65-dsRed, with very similar
kinetics to those shown by cells expressing EGFP-GR activated with
dexamethasone. By determining the half time of p65-dsRed nuclear occupation,
we have shown that the time of nuclear occupation is significantly reduced by
overexpression of EGFP-GR, by pre-stimulation with dexamethasone/RU486 or by a
combination of EGFP-GR expression and dexamthasone/RU486 treatment compared to
control cells expressing EGFP (Fig.
3C).
Effect of activation of the STAT6 pathway on the NF-B
pathway
Activated, overexpressed STAT6 has previously been shown to inhibit the
TNF-induced transcription of a synthetic NF-
B promoter
(Ohmori and Hamilton, 2000
).
We therefore wished to establish the nature and mechanism by which STAT6
activation by IL-4 could inhibit activation of the NF-
B pathway.
Pretreatment of HeLa cells expressing the control EGFP with IL-4 gave rise to
significant inhibition of subsequent TNF
induced NF-
B-directed
gene expression (Fig. 4). The
expression of EGFP-STAT6 fusion protein gave rise to a significant increase in
the IL-4-mediated inhibition of TNF
-stimulated gene expression. The
IL-4 mediated inhibition of TNF
-stimulated luciferase expression could
be overcome by overexpression of p65-EGFP and this was also true in cells
co-expressing EGFP-STAT6. Expression of EGFP-STAT6 also lowered the basal
transcription level from NF-
Bluc (data not shown). This suggests that
EGFP-STAT6 behaves in a similar manner to native STAT6 in its effect on the
NF-
B pathway.
|
We therefore applied imaging of the dynamics of p65 translocation to
investigate the mechanism of inhibition of activated STAT6 inhibition of the
NF-B pathway. Cells expressing p65-dsRed and either EGFP-STAT6 or EGFP
(control) were treated with TNF
either with or without IL-4
pretreatment. There was marked inhibition of p65-dsRed translocation by
expression of EGFP-STAT6 (Fig.
5A,B). Pre-stimulation with IL-4 inhibited p65-dsRed translocation
in cells that were overexpressing both EGFP (as a control) and EGFP-STAT6. The
effects of IL-4 and EGFP-STAT6 were also cumulative. Statistical analysis
(ANOVA) of the kinetics of p65-dsRed translocation under these conditions
showed that the maximal nuclear translocation of p65-dsRed was significantly
reduced by activation and/or overexpression of EGFP-STAT6
(P<0.05).
|
Effect of activation of the GR or STAT6 signalling pathways on the
dynamics of IB degradation in response to TNF
IB
is known to shuttle between the cytoplasm and nucleus, and
is believed to act as a chaperone to relocalize NF-
B to the cytoplasm.
Therefore, we investigated whether GR or STAT6 affected the timing of
I
B
degradation or localization. Transfection of cells with
I
B
-EGFP allowed us to investigate whether the increased nuclear
export of p65 by activated GR was due to stabilisation of I
B
after treatment with TNF
. For these studies, we used cells expressing
both p65-dsRed and I
B
-EGFP, because we have previously shown
that the ratio of the two proteins significantly affects the degradation rate
of I
B
-EGFP (Nelson et al.,
2002a
). There was no noticeable effect of preincubation with 10 nM
dexamethasone on resting cellular I
B
-EGFP localization, and
there was no significant effect upon the degradation rate of
I
B
-EGFP following treatment with TNF
(Fig. 6A).
|
We also investigated the effect of IL-4 stimulation of the STAT6 pathway
upon IB
-EGFP. In cells transfected with I
B
-EGFP
and p65-dsRed, we saw no effect of preincubation of IL-4 upon either p65 or
I
B
(data not shown). Subsequent stimulation with TNF
showed a marked reduction in the level of I
B
-EGFP degradation,
suggesting that pre-activation with IL-4 inhibited this step in the
NF-
B pathway (Fig. 6A).
This demonstrated a clear distinction between the mechanisms of inhibition of
the NF-
B pathway by STAT6 and GR.
We further analysed the IL-4-mediated inhibition of IB
by
investigating the effect of IL-4 upon phosphorylation of I
B
.
Western blotting demonstrated transient I
B
serine-32
phosphorylation after stimulation with 10 ng ml-1 TNF
.
Incubating cells with 10 ng ml-1 IL-4 for 40 minutes prior to
TNF
treatment caused a decrease in I
B
serine-32
phosphorylation, with very little phosphorylation 10 minutes post TNF
treatment (Fig. 6B).
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Discussion |
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RU486 is a type-II GC agonist that allows nuclear translocation of GR and
DNA binding but is not an efficient activator of transcription, because it
recruits the co-repressor NCoR rather than the co-activator p160 to GR
(Schulz et al., 2002).
Interestingly, RU486 treatment has been previously shown to inhibit
NF-
B directed transcription but is less potent than dexamethasone
(Heck et al., 1994
;
Heck et al., 1997
). We show
that preincubation with RU486 diminished the time of nuclear occupancy by a
similar extent to dexamethasone treatment in cells expressing EGFP-GR
(Fig. 3C). This observation
therefore suggests that the stimulation of the nuclear export of p65 does not
require the GR to adopt a transactivation competent conformation.
IB
is believed to control the nuclear/cytoplasmic status of
NF-
B, by shuttling into the nucleus, where it is thought to bind to the
NF-
B complex, masking the NLS so that the NES of I
B
predominates, resulting in cytoplasmic NF-
B/I
B. We investigated
whether GR might be increasing the rate at which I
B can export
NF-
B. This could be through increasing nuclear shuttling of I
B
or stabilization of I
B by preventing its phosphorylation by IKK and
subsequent degradation. Stimulation of cells expressing I
B
-EGFP
and p65-dsRed with dexamethasone showed no detectable alteration in the
localization of I
B
-EGFP (data not shown). Subsequent stimulation
with TNF
showed no apparent difference in the rate of
I
B
-EGFP degradation compared with the control
(Fig. 6A), suggesting that the
effect of GR upon nuclear export was not directed through I
B
.
Newly synthesized I
B
is also unlikely to be the target of
GR-mediated p65 export, as the rate of nuclear export after TNF
stimulation is too fast for new protein synthesis.
It therefore seems most likely that transrepression of p65 by GR either
occurs through direct interaction with p65 or via alternative proteins. It has
recently been demonstrated that other upstream proteins of the NF-B
signalosome, namely IKK
and NIK also shuttle in resting cells
(Birbach et al., 2002
). It is
likely that these contribute to the basal state of NF-
B and so are
potential targets for GR. However, because GR can bind p65
(Caldenhoven et al., 1995
;
Nissen and Yamamoto, 2000
),
this seems to be a more likely route of inhibition. Exactly how GR increases
p65 export remains unclear, however, because EGFP-GR appears to remain in the
nucleus when p65-dsRed is exported. GR-bound p65 might be a better target for
chaperones mediating CRM-1 dependent nuclear export and hence increasing p65
nuclear export. Another possibility is that GR inhibits another protein
interaction with p65 that would otherwise stabilize its accumulation in the
nucleus. One such interaction would be with the catalytic subunit of protein
kinase A (PKAc), which has been implicated as a member of the NF-
B
signalosome complex and shown to phosphorylate p65
(Zhong et al., 1997
).
Furthermore, PKAc overexpression has been shown to increase
NF-
B-mediated transcription, which could be inhibited by also
expressing GR (Doucas et al.,
2000
). Because GR overexpression does not alter p65 import rates,
it might alternatively be inhibiting phosphorylation of p65 by PKAc and
therefore decreasing its transcription initiation efficacy. This would rely
upon p65 nuclear import being independent of the phosphorylation state of p65,
which would fit with the findings of Nissen and Yamamoto
(Nissen and Yamamoto, 2000
),
who showed that GR bound p65 but did not affect DNA binding by p65. Instead,
GR inhibited the phosphorylation of the C-terminal domain of RNA polymerase
(pol) II in the transcription preinitiation complex (PIC), which is required
for transcription initiation. It is possible that p65 would have a lower
binding affinity for inactive PIC, and this would cause p65 dissociation in
the nucleus. Subsequently, the unbound nuclear p65 is more likely to be
targeted for nuclear export by I
B
. Both scenarios (GR inhibition
of cytoplasmic p65 phosphorylation and nuclear GR inhibition of RNA pol II
phosphorylation) can be combined by envisaging that the conformation of
phosphorylated p65 encourages the phosphorylation of RNA pol II in the
p65-bound PIC. In such a case, GR inhibition of RNA pol II phosphorylation and
increased nuclear export of p65 would both be seen as downstream effects of
the phosphorylation state of p65.
Overexpression of EGFP-STAT6 had a similar effect to GR on the NF-B
pathway. We have shown inhibition of NF-
B-mediated transcription by
IL-4 activated EGFP-STAT6 (Fig.
4). Also, investigation of single living cells expressing
EGFP-STAT6 and p65-dsRed showed marked inhibition of p65 translocation by both
activating the STAT6 pathway and overexpressing EGFP-STAT6
(Fig. 5). The observed
inhibition of p65-dsRed translocation was far greater than the transcriptional
inhibition from NF-
B-luc. This is expected because very little
transcription factor is required in the nucleus to activate transcription,
whereas fluorescence imaging of translocation shows gross properties of the
fusion protein. The effects of EGFP-STAT6 and IL-4 upon p65-dsRed
translocation were cumulative, suggesting that the inhibition was directed
through activated STAT6. However, the half-life of p65 nuclear occupation was
not affected to the same extent by activation of EGFP-STAT6 (not significant
compared with EGFP; data not shown) compared with activation of EGFP-GR.
EGFP-STAT6 slowed the rate of p65 translocation into the nucleus and the
overall maximal nuclear translocation was reduced by EGFP-STAT6
(Fig. 6A), whereas this was not
the case for EGFP-GR. Therefore, the modes of action appear to be different,
with IL-4-mediated inhibition minimizing p65 import and dexamethasone
increasing the rate of p65 nuclear export. Because EGFP-STAT6 appeared to be
blocking p65 import, we also investigated whether IL-4 had an effect upon
I
B
by monitoring I
B
-EGFP degradation in cells
pretreated with IL-4. There was significant inhibition of TNF
-mediated
I
B
-EGFP degradation (Fig.
6A), which was shown to correlate with a shorter time course of
native I
B
phosphorylation at serine 32, suggesting IL-4-mediated
inhibition of I
B
phosphorylation rather than ubiquitination
(Fig. 6B). This would explain
the block in p65 import because, in resting cells, p65 is held inactive by the
I
B
. It is possible that cytoplasmic STAT6 is responsible for
this inhibition, because activated STAT6 translocates to the nucleus slowly
(Nelson et al., 2002b
). A
recent report has also shown that activated STAT6 inhibits I
B
phosphorylation, supporting the current data
(Wei et al., 2002
). However,
inhibition of I
B
phosphorylation and degradation does not
entirely explain why mutation of the STAT6 DNA binding domain knocks out STAT6
suppression of NF-
B-mediated transcription
(Ohmori and Hamilton, 2000
).
One possibility is that STAT6 interacts directly with p65 and that this
interaction both blocks the binding of I
B
with p65 and masks the
NLS of p65. I
B
appears to be subject to phosphorylation by only
the IKK and/or proteasome-mediated degradation in the context of
I
B
/NF-
B dimers (Zandi
et al., 1998
; Nelson et al.,
2002a
) and therefore the rate of I
B
degradation
would be diminished in this model, whereas p65 nuclear import would also be
inhibited by the STAT6 interaction. Therefore, a direct interaction between
p65 and activated STAT6 could explain these observations.
We have investigated how GC and STAT6 pathways inhibit NF-B and have
shown them to work through different mechanisms. Both appear to occur
independently of up-or downregulation of transcription and also before
NF-
B bound the transcription initiation complex. These data suggest
complex interactions between signalling pathways affecting the cellular
equilibrium of transcription in resting cells and in cells responding to the
complex cocktail of cytokines present in vivo.
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Acknowledgments |
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