Interleukin-2 Inhibits Glucocorticoid Receptor Transcriptional Activity through a Mechanism Involving STAT5 (Signal Transducer and Activator of Transcription 5) but Not AP-1
Armelle Biola,
Philippe Lefebvre,
Mallory Perrin-Wolff1,
Marie Sturm,
Jacques Bertoglio and
Marc Pallardy
INSERM U461 (A.B., M.S., J.B., M.P., M.P.-W.) Faculté de
Pharmacie Paris-Sud 92296 Châtenay-Malabry, France
INSERM U459 (P.L.) Faculté de Médecine Henri
Warembourg 59045 Lille, France
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ABSTRACT
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Cytokines and glucocorticoids (GCs) signaling
pathways interfere with each other in the regulation of apoptosis and
gene expression in the immune system. Interleukin-2 (IL-2), through the
Janus kinase/signal transducers and activators of transcription
(Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways,
activates STAT5 and activated protein-1 (AP-1) transcription
factors, respectively, which are known to repress glucocorticoid
receptor (GR) activity, at least in part, through protein-protein
interactions. In this work, we have analyzed the mechanisms whereby
IL-2 down-regulates the GC-induced transactivation of the mouse mammary
tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T
lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5
binding site (-923/-914) was not required for IL-2-mediated
inhibition but identified both glucocorticoid response elements (GREs)
and the -104/+1 region as critical elements for this negative
response. The DNA binding activities of transcription factors required
for GC-mediated activation of the MMTV-LTR promoter and that bind to
the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2
treatment. Overexpression of wild-type STAT5B enhanced the effect of
IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B
(Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1
activation had no effect in this system. Direct interaction between
liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5
phosphorylation-independent manner. Overexpression of nuclear
coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor
coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that
the STAT5-repressive activity on the GC-dependent transcription may
involve direct interaction of STAT5 with GR, is dependent on the
promoter context and STAT5 activation level, and occurs independently
of coactivators levels in T cells.
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INTRODUCTION
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Glucocorticoids (GC) exert their biological effects through an
interaction with the glucocorticoid receptor (GR), a ligand-activated
transcription factor belonging to the nuclear receptor family. Two
isoforms of human GR have been described: GR
and GRß. GR
is
mainly located in the cytoplasm of unstimulated cells as part of a
large multiprotein inactive complex with heat shock proteins and
immunophilins. Hormone binding causes dissociation of this complex and
translocation of the receptor into the nucleus. Then, GR stimulates
hormone-dependent transcription through binding to a 15-bp
glucocorticoid responsive element (GRE) present in the regulatory
regions of responsive genes (1, 2). The function of GRß as a dominant
negative form of GR is still a matter of debate (3, 4). The mouse
mammary tumor virus (MMTV) promoter is one of the most studied
GC-responsive promoters. Its transcriptional activation requires
binding of GR to a cluster of four glucocorticoid response elements
(GREs) located within the U3 region of the long terminal repeat (LTR)
(5, 6). The promoter also contains binding sites for ubiquitous
transcription factors such as nuclear factor-1 (NF-1), octamer
transcription factors-1 and -2 (Oct-1 and Oct-2), and for other unknown
tissue-specific regulatory factors controlling the expression of MMTV
(7, 8, 9, 10). When stably transfected into mammalian cells and before
stimulation, the MMTV-LTR is reproducibly packaged into a phased array
of nucleosomes preventing NF-1 access to its binding site. Hormone
binding initiates chromatin remodeling, and the promoter becomes
accessible to NF-1. On transiently transfected MMTV-LTR plasmid DNA,
NF-1 binding occurs constitutively and is not affected by GR loading
(11).
To mediate their effects, steroid hormones and cytokines activate
various signaling pathways through intracellular or membrane receptors,
respectively. In immune cells, cross-talks between these signaling
pathways affect fundamental cellular processes such as proliferation,
differentiation, or apoptosis. Indeed, GCs suppress interleukin-4
(IL-4)-induced proliferation of the murine CTLL-2 cytotoxic T cell
line, without affecting the IL-2-driven growth of these cells (12). In
mouse T helper (Th) cell lines, the synthetic GC dexamethasone (DEX)
completely inhibits IL-2-induced cell proliferation, reduces
IL-4-mediated cell growth, and has no effect on IL-9-response (13). Rat
CD4+ T cells transiently exposed in vitro to DEX display an
altered pattern of cytokine production and develop a Th2 response (14).
IL-2 has a protective role against GC-induced cell death on T cell
hybridomas (15) and T lymphocytes (16, 17, 18). Moreover, IL-4 protects Th2
cells from DEX-induced apoptosis and IL-2 rescues Th1 cells from the
cytolytic effect of GCs, indicating that mature T cells can be saved by
their own growth factor (19). IL-9 is a potent inhibitor of GC-induced
apoptosis in thymic lymphoma cell lines (20). However, mechanisms
underlying interactions between GCs and cytokines signal transduction
pathways in immune cells remain poorly understood.
GR can establish protein-protein interactions, independently from
DNA-binding, with other transcription factors such as activated
protein-1 (AP-1), NF-
B, STAT-3 (signal transducer and activator of
transcription 3), and STAT-5 (21). These transcription factors are
activated by cytokines, resulting in positive or negative regulation of
GC-induced transcription. The outcome of cytokine stimulation on
GC-mediated transcription is dependent on the promoter context and on
the cell type. Indeed, in CEM, S49, and Jurkat lymphoid T cell lines,
phorbol myristate acetate (PMA), through induction of AP-1, enhances
DEX-induced transactivation of the MMTV-LTR, whereas it displays an
inhibitory effect in NIH 3T3 fibroblasts (22). In immune cells, GCs
inhibit NF-
B activation induced by tumor necrosis factor-
(TNF
), and GR was shown to physically interact with NF-
B, thereby
preventing its binding to DNA (23, 24). Furthermore, GCs were shown to
induce the expression of the inhibitory protein I
B
, trapping
NF-
B as an inactive complex in the cytoplasm (23, 24).
Overexpression of STAT-5 in PRL-activated COS-7 cells results in an
increased activity of the ß-casein gene promoter upon treatment with
GCs, whereas MMTV-LTR promoter activity was decreased under similar
conditions (25).
The aim of this work was to evaluate the effect of IL-2 on GR
transcriptional activity in lymphoid cells. We show that IL-2 strongly
inhibits GC-induced transcription from the MMTV promoter, whereas IL-2
alone weakly stimulates its activity. These effects were observed in
cells stably or transiently transfected with MMTV-LTR-luciferase
(MMTV-LTR-luc) constructs. We also demonstrate that IL-2 does not
impede binding of NF-1 or Oct-1 to their specific DNA binding sites. A
STAT5 binding site was identified in the 5'-region of the
promoter, which proved to be responsible for the positive regulation of
the promoter by IL-2. However, deletions and mutations within the MMTV
promoter showed that neither the STAT5-responsive element, nor other
uncharacterized DNA sequences, play a role in IL-2 inhibition of
MMTV-LTR-luc transactivation. AP-1 is not involved in the inhibitory
mechanism of IL-2 as shown by PMA treatment. In CTLL-2 cells
overexpressing STAT5B, IL-2 inhibition of GC-induced transcription is
enhanced when compared with normal cells, whereas overexpression of a
dominant negative form of STAT5B (Y699F) abolishes the IL-2- inhibitory
effect. Coimmunoprecipitation experiments indicated a physical
association between GR and STAT5, occurring in CTLL-2 cells treated
with DEX, or DEX and IL-2. Taken together, these results strongly
suggest a role for IL-2-induced STAT5 in the inhibition of GR
transcriptional activity in lymphocytes. The mechanism of inhibition
does not rely on a competition for limiting amounts of CBP
(CREB-binding protein) or SRC-1a (steroid receptor coactivator-1a),
since overexpression of the coactivators increases the IL-2 inhibitory
effect.
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RESULTS
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IL-2 Inhibition of GC-Mediated Transcription Is Promoter Context
Dependent
CTLL-2, a murine T cell clone depending on IL-2 for its growth,
was used to examine the effect of IL-2 on GC-induced transcriptional
activation of MMTV-LTR. CTLL-2 cells were transiently transfected with
the MMTV-LTR-luc plasmid, which contains the full-length MMTV LTR.
Cells were treated for 12 h with 100 nM DEX, 1 ng/ml
IL-2, or DEX and IL-2 (Fig. 1A
). IL-2
alone caused a modest enhancement of the luciferase activity in the
absence of DEX. In the presence of DEX, a 12-fold induction of the
luciferase activity was observed. Incubation of the cells with DEX and
IL-2 resulted in more than 70% inhibition of the transcriptional
activity of the promoter when compared with DEX-only conditions.

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Figure 1. Effect of IL-2 on GR Transcriptional Activity in
CTLL-2 Cells
A, IL-2 inhibits DEX-induced MMTV-LTR activity. CTLL-2 cells were
transiently transfected with MMTV-LTR-luc plasmid and stimulated for
12 h with 1 ng/ml IL-2, with or without 100 nM DEX, or
left untreated. Fold induction was calculated as the ratio of arbitrary
luciferase units in cells treated with IL-2, DEX, or DEX and IL-2
compared with untreated cells. Fold induction (DEX treated cells)
= (DEX activity - basal activity)/basal activity. Fold induction
(DEX + IL-2 treated cells) = (DEX + IL-2 activity - IL-2
activity)/basal activity. The value 1 was consequently affected to the
basal level of nontreated cells (NT). Results were obtained from two
independent experiments. SEM is indicated by error bars. B,
Effect of IL-2 on DEX-induced GRE5-EBV-TATA-CAT
transactivation. CTLL-2 cells were transiently transfected with the
GRE5-EBV-TATA-CAT plasmid driving the CAT reporter gene.
Cells were stimulated for 8 h with 1 ng/ml IL-2, with or without
100 nM DEX, or left untreated. Percentage of
chloramphenicol conversion represents the ratio between acetylated
chloramphenicol and total chloramphenicol (acetylated and
nonacetylated). A representative experiment out of three is shown here.
C, Effect of IL-2 on GR DNA binding activity. The DNA-binding activity
of in vitro translated recombinant rat GR was assayed by
EMSA after DEX activation. CTLL-2 cells were deprived of IL-2 for
3 h and treated for 1 h with IL-2 (1 ng/ml) or left untreated
(NS). Nuclear extracts were then prepared and used for competition experiments with
GR for DNA binding (1 µg, 2.5 µg, 5 µg). The DNA binding
specificity was determined by competition with a 50-fold excess of
either a cold GRE probe, or a random probe (Rd) and by supershift
experiments with 1 µg of anti-GR antibody (M20, Santa Cruz Biotechnology, Inc.) or of control antibody (C). The first lane
corresponds to a nonprogrammed reticulocyte lysate. NS, Not stimulated.
A representative experiment out of four is shown here.
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We then selected a simple promoter, composed of five synthetic
palindromic GREs adjacent to a minimal TATA box
(GRE5-EBV-TATA-CAT), to evaluate whether the
mechanism of the IL-2 inhibitory effect was mediated through GREs.
CTLL-2 cells were thus transiently transfected with this reporter
plasmid and incubated for 8 h in the presence or in the absence of
DEX (100 nM), with or without 1 ng/ml IL-2. No inhibition
of the DEX-induced promoter activity was observed in the presence of
IL-2. Rather, IL-2 slightly enhanced DEX-induced
GRE5-EBV-TATA-CAT transactivation (Fig. 1B
).
These results suggest that the IL-2 inhibitory effect on GC-stimulated
MMTV promoter activity is probably promoter context dependent and is
not uniquely mediated through GREs.
We assessed the effect of IL-2 on GR DNA-binding activity by EMSA
(electrophoretic mobility shift assay) using in vitro
translated recombinant GC receptor (Fig. 1C
). At least two major
complexes with different mobilities were detected, despite the unique
GRE sequence of the DNA probe. This could result from a partial
proteolysis of the GR in the rabbit reticulocyte lysate. These
complexes bound specifically to the GRE DNA-probe, since their
formation was competed by a 50-fold molar excess of unlabeled GRE DNA
probe, but not by the nonspecific random DNA fragment. Furthermore,
addition of the specific anti-GR antibody to the binding reaction did
not block complex formation, but generated an antibody-protein-DNA
ternary complex resulting in a further reduction of the mobility of all
the protein-DNA complexes.
Competition experiments were performed with increasing amounts (1, 2.5,
and 5 µg) of nuclear extracts of CTLL-2 cells deprived of IL-2 for
3 h and treated for 1 h with IL-2 (1 ng/ml), or left
untreated. Consistent with the results presented in Fig. 1B
, IL-2
treatment did not impede the binding of GR to DNA (Fig. 1C
) since GR
DNA-binding activity was similar in the presence of nuclear extracts
from nontreated or IL-2-treated cells. Addition of nuclear extracts did
not generate lower-mobility complexes, which could result from the
interaction of DNA-bound GR with other cellular proteins, induced or
not by IL-2. These results suggest that IL-2 stimulation of T cells
does not unmask activities able to perturb GR binding to DNA.
IL-2 Inhibits DEX-Induced MMTV-LTR Activity of Stably Transfected
Templates
Modifications of chromatin structure are known to affect
GC-induced transcriptional activity of the MMTV promoter. IL-2 could
interfere with chromatin remodeling by GR, thus affecting the
activation of transcription. We therefore compared stably integrated
templates and transiently transfected templates, which have been shown
to display, or not, a chromatin architecture, respectively, for the
effect of IL-2 on GC-induced transcriptional activity of MMTV-LTR. To
this end, we stably transfected the wild-type MMTV-LTR-luc plasmid in
CTLL-2 cells. Several clones were isolated and tested for their
responsiveness to GC treatment. All clones tested responded similarly
to IL-2 and GC stimulation, albeit with a different amplitude, which is
likely to be related to the number of integrated copies. The clone
showing the highest inducibility of GC-dependent transactivation
(CTLL-2 pLTR 2E) was selected for further experiments. Results
presented in Fig. 2
show that treatment
of these cells with DEX for 12 h results in a 20-fold increase in
luciferase activity when compared with the basal transcription level.
Simultaneous addition of IL-2 and DEX results in an IL-2 dose-dependent
inhibition of DEX induction of the MMTV promoter activity. At a
saturating dose of 1 ng/ml of IL-2, the promoter activity is almost
completely abolished (91% inhibition) and reaches basal level measured
in the absence of hormone stimulation. As previously observed with
transiently transfected templates, IL-2 alone positively regulated
MMTV-LTR activity. These results obtained with the chromatin templates
are thus comparable to the transiently transfected ones, suggesting
that the IL-2 effect is not affected by integration of the promoter
into chromatin.

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Figure 2. IL-2 Inhibits GC-Induced MMTV-LTR-luc
Transactivation in Stably Transfected CTLL-2 pLTR Cells
CTLL-2 cells stably transfected with MMTV-LTR-luc were washed three
times to remove IL-2, and then stimulated for 12 h with either 0.2
ng/ml, 0.5 ng/ml, or 1 ng/ml IL-2, with or without 100 nM
DEX, or left untreated. Fold induction was calculated as in Fig. 1A .
Data from a representative experiment are expressed as a mean of
triplicates. SEM is indicated by error bars.
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MMTV-LTR Contains a STAT5 Binding Site That Is Not Involved in IL-2
Inhibition
Compared with the simple synthetic promoter of the
GRE5-EBV-TATA-CAT plasmid, the composite nature
of the MMTV promoter (Fig. 3
) therefore
raised the hypothesis of the existence of one (or more) regulatory
element (s) necessary for the inhibitory effect of IL-2. Internal
deletions of the MMTV LTR (-967 to -220, and -860 to -220) allowed
us to localize a STAT5 consensus binding site
(TTCGGAGAA) (Fig. 3
).

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Figure 3. Structure of the MMTV-LTR Promoter
Numbers indicate the position relative to the transcriptional
initiation site (+1). NRE, Negative regulatory element; HRE, hormone
responsive element; STAT5 RE, STAT5 responsive element; AA and A,
regulatory elements (44 , 46 ).
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IL-2 activates the Janus family tyrosine kinases Jak1 and Jak3,
resulting in subsequent tyrosine phosphorylation and DNA binding of
STAT5 (26, 27). We first characterized the IL-2-mediated activation of
STAT5 in CTLL-2 cells using transcriptional activation assays (data not
shown) and EMSA. No STAT5-DNA binding activity was present in CTLL-2
cells deprived of IL-2 for 3 h (Fig. 4A
). However, after 1 h of IL-2
stimulation (1 ng/ml), a strong STAT5-DNA binding was observed
(Fig. 4A
). Supershift experiments showed that STAT5A and STAT5B were
activated in CTLL-2 cells upon IL-2 treatment. We then evaluated by
EMSA whether IL-2-activated STAT5 proteins could bind to the
STAT-responsive element of the MMTV promoter (-923/-914) (Fig. 4B
). A
DNA-bound complex was detected in IL-2-treated cells and could be
completely supershifted with the anti-STAT5 serum (Fig. 4B
), suggesting that STAT5 was the only
component of the complex formed on the STAT/MMTV probe in CTLL-2 cells.
To assess the functional activity of this STAT5 element, we mutated
this sequence to disrupt STAT5-DNA binding on the MMTV-LTR distal
regions of the pLTR-luc and the p-1,180/-860-luc plasmids to generate
pLTRmut-luc and p-1180/-860 mut-luc plasmids. CTLL-2 cells were then
transiently transfected with wild-type or mutated constructs and
treated for 12 h with or without IL-2 (1 ng/ml). Results presented
in Fig. 4C
show that mutation of the STAT5 site dramatically reduces
the basal activity of the promoter measured in untreated cells. In
addition, the activity of the mutated constructs was not significantly
affected by IL-2, strongly suggesting that this site plays a role in
the positive regulatory effect of the cytokine on the MMTV promoter
activity. We then investigated whether this STAT5-binding site could
play a role in the IL-2-inhibitory effect on DEX-induced MMTV-LTR
transactivation. To this end, CTLL-2 cells were transiently transfected
with the pLTR-luc and the pLTRmut-luc plasmids and treated for
12 h with IL-2 (1 ng/ml), with or without DEX (100
nM), or left untreated. Results show that the inducibility
of the mutated promoter is enhanced upon DEX treatment compared with
the wild-type promoter, due to the reduced level of the basal activity.
Nevertheless, the effect of IL-2 measured with pLTR-luc and pLTRmut-luc
(84% and 75%, respectively) is comparable with the two constructs
(Fig. 4D
), suggesting that the STAT5-binding site does not contribute
to the IL-2-inhibitory effect.

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Figure 4. Influence of the STAT5 Binding Site on IL-2
Inhibition of DEX-Induced MMTV-LTR Activity
A, Activation of STAT5A and STAT5B by IL-2 in CTLL-2 cells. Nuclear
extracts were prepared from CTLL-2 cells after 3 h of IL-2
deprivation and 1 h of treatment with or without IL-2 (1 ng/ml).
Competition experiments were performed in the presence of a 25-fold
excess of either a cold GAS probe, or a random probe (Rd). For
supershift experiments, 1.5 µl of the preimmune serum (PIS) or of the
specific anti-STAT5A or anti-STAT5B sera were preincubated with nuclear
extracts for 2 h at 4 C before addition of probe. B, Binding of
STAT5 on the STAT response element (STAT RE) of the MMTV-LTR promoter.
Nuclear extracts were prepared as described in panel A. Competition
experiments were performed in the presence of a 25-fold excess of
either a cold STAT/MMTV probe, or a random probe (Rd). For supershift
experiments, 1 µl of the preimmune serum (PIS) or of the anti-STAT5
serum were preincubated with nuclear extracts for 2 h at 4 C
before addition of probe. C, Effect of the mutation of the STAT5 RE on
both the basal and IL-2-induced activity of the MMTV promoter. CTLL-2
cells were transiently transfected with the pLTR-luc, pLTRmut-luc,
p-1180/-860-luc, and p-1180/-860 mut-luc plasmids and stimulated for
12 h with 1 ng/ml IL-2, or left untreated. Results are expressed
in arbitrary luciferase units. Results of a representative experiment
out of three. NT, Nontreated. D, Effect of the mutation of the STAT5 RE
on the IL-2 inhibition of the DEX-induced MMTV promoter
activity. CTLL-2 cells were transiently transfected with the pLTR-luc
or the pLTRmut-luc plasmids and stimulated for 12 h with 1 ng/ml
IL-2, with or without DEX, or left untreated. Fold induction was
calculated as in Fig. 1A . Data from a representative experiment are
expressed as a mean of duplicates. SEM is indicated by
error bars.
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Influence of Deletions within the MMTV-LTR on IL-2 Inhibition
We performed 5'-end deletions of the promoter up to positions
-325, -223, -200, and -104 (Fig. 5A
)
to identify cis-acting sequences along the MMTV promoter
involved in the IL-2-mediated inhibition. CTLL-2 cells were transiently
transfected with these constructs and cultured for 12 h in the
presence or in the absence of 100 nM DEX and/or
IL-2 (1 ng/ml). Results show that the p104-luc plasmid, which lacks the
two distal GREs, does not retain any hormone inducibility. Sequences
located upstream of the -200 position can be deleted without impairing
the hormone response (Fig. 5B
). The responsiveness of p325-luc,
p223-luc, and p200-luc plasmids to DEX stimulation is inhibited by
56%, 65%, and 62% upon IL-2 treatment (1 ng/ml), respectively. The
p223-luc and p200-luc deletion mutants show an increased GC-induced
transcriptional activity compared with the MMTV-LTR-luc plasmid,
suggesting that upstream negative regulatory sequence(s) were removed
(Fig. 5B
).

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Figure 5. Influence of Deletions within the MMTV-LTR on IL-2
Inhibition of GC-Induced Transcription in Transiently Transfected
CTLL-2 Cells
A, Schematic representation of the MMTV-LTR deletion mutants driving
the luciferase reporter gene. LUC, luciferase. Numbers
indicate the position relative to the transcriptional initiation site
(+1). B, Effect of IL-2 on the DEX-induced transactivation of MMTV-LTR
deletion mutants. CTLL-2 cells were transiently transfected with 10
µg of the different plasmids and were then stimulated for 12 h
with 1 ng/ml IL-2, with or without 100 nM DEX, or left
untreated. Fold induction was calculated as in Fig. 1A . Inductions were
calculated from two independent experiments. SEM is
indicated by error bars. NT, Not treated. C, Effect of
IL-2 on the DEX-induced transactivation of p2GRE-104-luc plasmid.
CTLL-2 cells were transfected as in panel B, except that the
p2GRE104-luc construct was used as a reporter gene. NT, Not treated.
Results from two independent experiments. SEM is indicated
by error bars.
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To investigate the role played by other functional
cis-acting elements located in the region spanning from
-200 to -104, we constructed the p2GRE104-luc plasmid (Fig. 5A
) by
substituting two consensus synthetic GREs for the sequence spanning
from the 5' end to the -104 position within the MMTV-LTR promoter.
Results from Fig. 5C
show that the basal luciferase activity of this
plasmid is very low and remains unaffected by IL-2. Very importantly,
addition of IL-2 reduced by 52% the DEX-induced transcriptional
activation of this promoter. These data demonstrate that the most
proximal region of the promoter, including the two proximal
hemi-palindromic GREs, the NF-1 and Oct-1 binding sites, as well as the
TATA box, are necessary and sufficient to convey the inhibitory effect
of IL-2.
IL-2 Does Not Impede NF-1 or Oct-1 Binding to Their Cognate DNA
Response Elements
Given that NF-1 binding to MMTV-LTR is an absolute prerequisite
for GC-induced MMTV transactivation, we evaluated whether IL-2 inhibits
NF-1 binding to its specific recognition site. We conducted EMSA using
a NF-1 probe whose sequence is similar to the NF-1 binding site present
in the MMTV promoter. Results from Fig. 6A
show that IL-2 does not reduce NF-1
binding activity.

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Figure 6. Effect of IL-2 and DEX on NF-1 or Oct-1 DNA Binding
Activity
Nuclear extracts were prepared from CTLL-2 cells after 3 h of IL-2
deprivation and 1 h of treatment with DEX (100 nM)
and/or IL-2 (1 ng/ml). A, NF-1 DNA binding activity. Competition
experiments were performed in the presence of a 25-fold excess of
either a cold NF-1 probe, or a random probe (Rd). B, Oct-1 DNA binding
activity. Competition experiments were performed in the presence of a
25-fold excess of either a cold Oct-1 probe, or a random probe (Rd).
For supershift experiments, 3 µg of the control IgG or of the
specific anti-Oct-1 IgG were preincubated with nuclear extracts for
2 h at 4 C before addition of probe.
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Oct-1 is a POU family transcription factor constitutively expressed in
many cell types, which specifically interacts with the octamer motif
ATGCAAAT. The MMTV promoter contains two functional octamer-related
elements involved in basal and steroid-induced transcription. Mutation
of both octamer motifs was shown to strongly reduce GC-induced MMTV
promoter activity (28). To determine the effect of IL-2 on Oct-DNA
binding activity, we performed EMSA experiments with an oligonucleotide
derived from MMTV sequence between -61 and -32 positions. Results
show that IL-2 does not significantly affect the binding of Oct
proteins to their specific DNA sequence (Fig. 6B
). To characterize the
nature of the Oct family member bound to the probe in CTLL-2 cells, we
performed supershift experiments with a specific anti-Oct-1 polyclonal
antibody directed against the carboxy terminus of the protein. The
anti-Oct-1 IgG, but not the control IgG, completely displaced the
specific complex for all tested treatments, indicating that Oct-1 is an
integral part of the complex formed on the Oct probe in CTLL-2
cells.
AP-1 Is Not Involved in the IL-2 Inhibition of GC-Induced
MMTV-LTR Transactivation
These results prompted us to determine which IL-2-dependent signal
transduction pathway was involved in the inhibition of GC-induced
transcription. The AP-1 transcription factor, which plays a crucial
role in cell cycle control and survival of lymphoid cells, has been
shown to physically interact with the GR, resulting in a mutual
transcriptional repression (29, 30, 31). IL-2 and PMA have been shown to
increase AP-1 DNA binding and transcriptional activities in CTLL-2
cells (Ref. 32 and Fig. 7A
). Results
showed that PMA did not repress the GR transcriptional activity
assessed with the MMTV-LTR-luc plasmid (Fig. 7B
). These results were
obtained in stably transfected CTLL-2 pLTR cells (Fig. 7B
), as well as
in transiently transfected CTLL-2 cells (data not shown).

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Figure 7. Effect of IL-2 and PMA on GC-Induced MMTV-LTR-luc
Transactivation in Stably Transfected CTLL-2 pLTR Cells
A, IL-2 and PMA activate AP-1. CTLL-2 cells were transiently
transfected with the PMA-inducible 5XTRE-tk-CAT plasmid driving the CAT
reporter gene. Cells were stimulated for 8 h with 1 ng/ml IL-2, or
50 ng/ml PMA, or left untreated. Percentage of chloramphenicol
conversion represents the ratio between acetylated chloramphenicol and
total chloramphenicol (acetylated and nonacetylated). Induction was
calculated as the ratio of percentage of chloramphenicol conversion in
cells treated with IL-2 compared with untreated cells (x5) or in cells
treated with PMA (x4) compared with untreated cells. A representative
experiment out of three is shown here. B, Effect of IL-2 and PMA on
DEX-induced MMTV-LTR transactivation. CTLL-2 cells stably transfected
with MMTV-LTR-luc were washed three times to remove IL-2, and then
stimulated for 12 h with 1 ng/ml IL-2, or 50 ng/ml PMA with or
without 100 nM DEX, or left untreated. Fold induction was
calculated as in Fig. 1A , except for DEX + PMA induction = (DEX +
PMA activity - PMA activity)/basal activity.
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Role of STAT5B in IL-2 Inhibition of GC-Induced MMTV-LTR
Transactivation
We demonstrated above that STAT5 DNA binding on the distal
region of the promoter could not account for the inhibitory
effect of IL-2. Since the proximal part of the promoter contains no
STAT5 binding site, we assessed whether STAT5 could alter the promoter
activity by engaging protein-protein interactions with other
transcription factors. For this purpose, we used two complementary
approaches. CTLL-2 cells were stably transfected with either the
wild-type form of STAT5B (clone 15A) or a mutated dominant negative
STAT5B protein under the control of a tetracycline-regulated (Tet-Off)
gene expression system (33). The dominant negative form of STAT5B
cannot be activated by IL-2 since it possesses one mutation on tyrosine
699 (Y699F). Both STAT5B proteins were expressed as fusion
proteins (Myc-STAT5B and Myc-STAT5B Y699F) with a Myc tag to screen
their expression independently of the endogenous form of STAT5B, using
the 9E10 anti-Myc antibody.
Overexpression of wild-type Myc-STAT5B protein was achieved after
48 h of culture in the absence of tetracycline. In the presence of
tetracycline (1 µg/ml), the expression of this protein was fully
repressed (Fig. 8A
, right
insert). Cells were then transiently transfected with the
p2GRE104-luc construct and stimulated for 12 h in the presence or
in the absence of 100 nM DEX and/or IL-2 (1 ng/ml
and 10 ng/ml). In STAT5B overexpressing cells, IL-2 (1 ng/ml)
inhibition of GC-induced p2GRE104-luc transactivation was enhanced
by almost 20% (54% inhibition in overexpressing STAT5B cells compared
with 36% inhibition in wild-type cells). This difference was not
detectable when a 10-fold higher concentration of IL-2 (10 ng/ml) was
used, leading to a maximal inhibition of DEX-induced p2GRE104-luc
transactivation (Fig. 8A
).

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Figure 8. Role of STAT5B in IL-2 Inhibition of GC-Induced
p2GRE104-luc Transactivation
A, Effect of wild-type STAT5B overexpression. CTLL-2 Myc-STAT5B 15 A
cells were cultured for 48 h in complete medium containing 1 ng/ml
IL-2, in the presence or in the absence of tetracycline (1 µg/ml).
Cells were then transiently transfected with 20 µg of the
p2GRE104-luc construct and stimulated for 12 h with either 1 ng/ml
or 10 ng/ml IL-2, with or without 100 nM DEX, or left
untreated. Results are expressed in percentage of IL-2 inhibition of
GC-induced p2GRE-104-luc transactivation. At the end of the incubation
period, Myc-STAT5B overexpression was characterized using the
monoclonal 9E10 anti-myc antibody (right inset). Results
of a representative experiment out of three. Tet, Tetracycline. B and
C, Expression of the STAT5B Y699F protein in clones 9, 4, and 5. CTLL-2
Myc-STAT5B Y699F cells (clones 4, 5, and 9) were cultured for 48 h
in complete medium containing 1 ng/ml IL-2, in the absence of
tetracycline. For all points, cells were deprived of IL-2 for 2 h
and stimulated for 12 h with 500 pg/ml IL-2. Whole cell lysates
were prepared and probed with the monoclonal 9E10 anti-myc antibody to
control Myc-STAT5B Y699F expression compared with untransfected CTLL-2
cells (C in panel B). The same extracts were assayed for STAT5B
activation using the DNA affinity precipitation method. Bound proteins
were probed with a specific anti-STAT5B antibody (panel C). Tet,
Tetracycline; C, CTLL-2 cells; WCE, whole-cell extracts. D, Effect of
Myc-STAT5B Y699F mutant overexpression. CTLL-2 Myc-STAT5B Y699F cells
(clones 4, 5, and 9) were cultured for 48 h in complete medium
containing 1 ng/ml IL-2, in the absence of tetracycline. Cells were
then deprived of IL-2 for 2 h, transiently transfected with 20
µg of the p2GRE104-luc construct, and stimulated for 12 h with
500 pg/ml IL-2, with or without 100 nM DEX, or left
untreated. Results are expressed in percentage of IL-2 inhibition of
GC-induced p2GRE-104-luc transactivation. Results from a representative
experiment out of two. E, Physical interaction between STAT5 and the
GR. CTLL-2 cells were deprived of IL-2 for 3 h and treated for
1 h with IL-2 (1 ng/ml) with or without 100 nM DEX, or
left untreated. Lysates were immunoprecipitated with an anti-GR
antibody (BuGR2, Affinity BioReagents, Inc.).
Immunocomplexes were separated by SDS-PAGE, transferred to PVDF
membrane, and detected with an anti-STAT5 antibody (upper
panel) or an anti-GR antibody (lower panel). IP,
Immunoprecipitation; IB, immunoblot. F, Physical interaction between
STAT5B Y699F and the GR. CTLL-2 Myc-STAT5B Y699F cells (clone 5) were
cultured for 48 h in complete medium containing 1 ng/ml IL-2, in
the absence of tetracycline. Cells were then deprived of IL-2 for
3 h, treated for 1 h with DEX (100 nM) and IL-2
(1 ng/ml). Lysates were immunoprecipitated with an anti-GR antibody
(BuGR2, Affinity BioReagents, Inc.). Immunocomplexes were
separated by SDS-PAGE, transferred to PVDF membrane, and detected with
an anti-myc antibody (9E10). WCE, Whole cell extract; IP,
Immunoprecipitation; IB, immunoblot.
|
|
Three stable cell lines (clones 4, 5, and 9) expressing different
levels of the dominant negative Myc-STAT5B Y699F protein were
established (Fig. 8B
), since no clone displaying high regulatable
levels of Myc-STAT5B Y699F could be obtained. Analysis of STAT5
activation in these clones, by DNA affinity precipitation with a
5'-biotinylated IFN
-activated sequence (GAS) oligonucleotide,
revealed that a dominant negative effect of STAT5B Y699F was only
achieved with clone 5, which showed the highest level of expression of
this protein (Fig. 8C
).
CTLL-2 Myc-STAT5B Y699F cells (clones 4, 5, and 9) were cultured
for 48 h in the absence of tetracycline. Cells were then
transiently transfected with the p2GRE104-luc construct and stimulated
for 12 h in the presence or in the absence of 100 nM
DEX and/or IL-2 (500 pg/ml). In clones 4 and 9, DEX-induced
p2GRE104-luc transactivation was still inhibited (-55% and -51%,
respectively) despite expression of the dominant negative form of
STAT5B (Fig. 8D
). These results were correlated with STAT5 activation
levels (Fig. 8C
). IL-2 inhibition of DEX-induced p2GRE104-luc
transactivation, however, was almost completely abolished (-6%) in
clone 5, which expresses the highest level of the Myc-STAT5B Y699F
protein (Fig. 8D
). These results showed that activation of STAT5 is a
key event in the inhibition of GC-induced MMTV transactivation by
IL-2.
We postulated that STAT5 might physically interact with the GR,
explaining the transcriptional interference existing between these two
factors. Coimmunoprecipitation experiments were performed with an
anti-GR antibody using whole-cell extracts from CTLL-2 cells deprived
of IL-2 for 3 h and treated for 1 h with IL-2 (1 ng/ml), DEX
(100 nM), DEX and IL-2 (1 ng/ml), or left untreated.
Blotting of the GR immunocomplex with an anti-STAT5 antibody allowed us
to detect coprecipitated STAT5 in DEX- or DEX and IL-2-treated cells
(Fig. 8E
). These results suggested that GR activation, but not STAT5
activation, is a prerequisite for GR-STAT5 complex formation. To
confirm this observation, we assessed whether STAT5B Y699F could
interact with the GR. For this purpose, CTLL-2 STAT5B Y699F cells
(clone 5) were cultured for 48 h in the absence of tetracycline,
deprived of IL-2 for 3 h, and then treated for 1 h with DEX
(100 nM) and IL-2 (1 ng/ml). Cell lysates were then
immunoprecipitated with an anti-GR antibody, and GR immunocomplexes
were separated by SDS-PAGE and probed with an anti-myc antibody.
Results presented in Fig. 8F
show that STAT5B Y699F physically
interacts with GR, confirming that STAT5B tyrosine phosphorylation is
not necessary for GR-STAT5 association.
Role of Coactivators in the IL-2 Inhibition of GC-Induced
MMTV-LTR Transactivation
Since CBP was described to interact with both the GR and
STAT5, we tested the hypothesis of a competition between GR and STAT5
for limiting amounts of the coactivator. CTLL-2 cells were transiently
transfected with p2GRE104-luc and pCMV-2N3T-CBP or pCMV-2N3T plasmids,
cultured for 48 h in complete medium containing 1 ng/ml IL-2, and
then treated for 12 h with DEX (100 nM), IL-2 (1
ng/ml), DEX and IL-2, or left untreated (Fig. 9A
). The transfection of increasing
quantities of CBP (1, 5, and 10 µg plasmid) resulted in an
enhancement of the IL-2 inhibitory effect on GC-induced p2GRE104-luc
transactivation (62%, 69%, and 72%, respectively, compared with 42%
with the control vector), ruling out the possibility of a squelching of
CBP by STAT5 as a mechanism of IL-2 inhibition (Fig. 9A
). Moreover, we
could determine that transfection of 1 µg pCMV-2N3T-CBP leads to an
enhancement of DEX-induced p2GRE104-luc transactivation, showing that
CBP participates in GC-induced transactivation. However, transfection
of 10 µg of plasmid has a negative effect on the promoter activity.
Taken together, these results suggest that CBP is a modulator of MMTV
promoter activity. We then evaluated the role of another GR
coactivator, SRC-1a, in the IL-2 inhibitory effect (Fig. 9B
).
Overexpression of SRC-1a causes a slight enhancement of the IL-2
inhibition of DEX-induced p2GRE104-luc transactivation (47%
vs. 35% for 5 µg of plasmid), indicating that the
mechanism of inhibition does not rely on a competition for limiting
amounts of SRC-1a (Fig. 9B
). Again, transfection of high amounts of
SRC-1a leads to a down-regulation of the MMTV promoter activity.

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Figure 9. Role of Coactivators in IL-2 Inhibition of
GC-Induced p2GRE104-luc Transactivation
A, Effect of CBP overexpression on the IL-2 inhibitory effect. CTLL-2
cells were transiently transfected with the p2GRE104-luc plasmid, and
pCMV-2N3T (control vector, 10 µg) or pCMV-2N3T-CBP (1, 5, or 10
µg). Cells were cultured for 48 h in complete medium containing
1 ng/ml IL-2 and then stimulated for 12 h with 1 ng/ml IL-2, with
or without DEX, or left untreated. Fold induction was calculated as in
Fig. 1A . Representative experiment out of three. NT, Not treated. B,
Effect of SRC-1a overexpression on the IL-2 inhibitory effect. CTLL-2
cells were transiently transfected with the p2GRE104-luc plasmid and
pCR3.1 (control vector, 5 µg) or pCR3.1-SRC-1a (0.5, 1, or 5 µg).
Cells were cultured for 48 h in complete medium containing 1 ng/ml
IL-2 and then stimulated for 12 h with 1 ng/ml IL-2, with or
without DEX, or left untreated. Fold induction was calculated as in
Fig. 1A . Results of a representative experiment out of three. NT, Not
treated.
|
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DISCUSSION
|
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Cross-talks between signal transduction pathways controlled by GCs
and cytokines are believed to play an important role in different
pathologies such as asthma or lymphoma (34). In addition, how cytokines
modulate GC-regulated genes is still a matter of debate in the field of
apoptosis (35). We and others have previously found that IL-2 was able
to inhibit GC-induced apoptosis in murine IL-2-dependent CTLL-2 cells
(17, 18, 32). In these lymphoid T cells, apoptosis provoked by GC is
dependent upon the transcriptional activity of GR (A. Biola,
unpublished data). IL-2 activates three major transduction pathways:
the phosphatidylinositol-3 kinase (PI3K) pathway, the Jak-STAT pathway
leading to STAT-3 and STAT-5 activation, and the Ras-MAPK
(mitogen-activated protein kinase) pathway resulting in AP-1 activation
(Refs. 36, 37 for review). In the present paper, we investigated the
mechanism underlying IL-2 inhibition of GC-induced transactivation of
MMTV-LTR. Since we were interested in evaluating this effect in
physiological conditions, we used murine lymphoid cells expressing
endogenous levels of the GR and of IL-2 receptor components and
transduction partners.
DEX-induced MMTV-LTR transcriptional activity was significantly reduced
upon IL-2 addition at saturating concentrations. However, GC-dependent
transactivation measured with the
GRE5-EBV-TATA-CAT plasmid (38) was not impaired
by IL-2, suggesting that this effect depends on the promoter context,
and that IL-2 did not prevent binding of DEX to the GR or translocation
of the GR to the nucleus. Moreover, GR DNA binding activity was not
affected in the presence of nuclear extracts of IL-2-treated CTLL-2
cells, showing that IL-2 did not induce the appearance of inhibitory
factors.
In the MMTV-LTR promoter, binding of the activated GR to GREs initiates
a remodeling of the chromatin that displaces nucleosome B and makes the
promoter accessible to NF-1, a step necessary for GC-dependent
transcription to occur. This mechanism is not observed on
chromatin-free templates (39, 11). Binding of the octamer motifs
in vivo was also observed to be strictly hormone dependent,
and Oct/GR interactions result in a transcriptional cooperativity
between these two factors (40). We observed that IL-2 inhibition was
still effective when MMTV-LTR was stably integrated into chromatin, and
that IL-2 did not alter NF-1 or Oct-1 binding capacity to their cognate
DNA sequences, indicating that inhibition does not occur through
modulation of either NF-1 or Oct-1 DNA binding activities.
Another mechanism that could account for IL-2 inhibition is the
activation of a trans-regulatory factor acting by binding to
the MMTV-LTR. Several sequences located within the U3 region of the
MMTV-LTR have been shown to regulate its activity (41). The MMTV
promoter contains four HREs (hormone responsive elements): two distal
palindromic sites (located between -184 and -114 positions) and two
proximal hemipalindromes (located between positions -98 and -78) (see
Fig. 3
). Deletion of the two distal GREs/HREs is sufficient to abolish
GC-induced transactivation (see Fig. 5B
) (42). A region around 1,090
to -900 within the MMTV-LTR has been delimited as an enhancer that
seems to be mainly involved in mammary specificity (41, 43). Deletion
of a regulatory element located between -294 to -200 termed AA
element has also been shown to decrease GC-induced transactivation
(42). Moreover, within this AA element a 20-bp region located between
-223 and -201 seems to play a regulatory role (44). At least three
negative regulatory elements have been described between -861 and
-364 (41). An AP-1 site has also been described between position -766
and -737 (45). We identified in this work a consensus STAT5-binding
site between positions -923 and -914 of the MMTV-LTR and showed the
involvement of this sequence in the IL-2 positive regulation of the
promoter activity, but not in the IL-2 inhibitory effect on DEX-induced
MMTV transactivation. This result rules out a role for a STAT5
DNA-binding element in the negative regulation of MMTV-LTR by IL-2.
Deletion of the entire 5'-end of the LTR up to the indicated position
allowed us to rule out the involvement of regions upstream of position
-325 (enhancer region, negative regulatory elements, AP-1 site),
between -325 and -223 and of the 20-bp sequence within the AA
element, respectively, in the IL-2-inhibitory effect. However, this
approach did not evaluate whether sequences located between GREs could
play a role. Indeed, a sequence located between the two distal GREs
(-163 to -147) and termed A element has been described to regulate
negatively MMTV-LTR activity upon binding of a
trans-negative modulator named C1 in 6.10.2 rat hepatoma
cells (46). However, data generated with the p2GRE-104 plasmid did not
confirm this hypothesis in CTLL-2 lymphocytes. Taken together, our
results obtained with these deletion mutants suggested that the
-104/+1 region is critical for the IL-2 negative response and that a
trans-acting IL-2-activated factor is not involved in the
regulation of MMTV-LTR activity by IL-2.
We then decided to identify which component of the IL-2 signal
transduction pathway was involved in the IL-2-inhibitory effect. This
approach could also clarify whether an IL-2-induced factor could
interact with GR-transcriptional activity without binding on the
DNA.
IL-2 leads to AP-1 activation in CTLL-2 cells (Ref. 32 and Fig. 7A
).
AP-1 and GR have been shown to mutually interfere with their
transactivating functions (29, 30, 31). Elevated c-Jun or c-Fos levels
can inhibit GR-dependent transcription from the MMTV-LTR promoter or
from promoters carrying only GREs (29, 30, 31). However, when CTLL-2 cells
were treated with PMA and DEX, no inhibition of MMTV-LTR
transactivation was found, although AP-1 activity was clearly induced
as assayed by gene reporter assays. We note that the composition of the
AP-1 complex could differ between IL-2 or PMA treatment (32),
explaining this result. Indeed, cell-specific factors and AP-1
composition may affect the outcome of the effect of AP-1 on
GR-transcriptional activity (22, 47, 48).
The Jak-STAT pathway has been recently described to interact with
GC-dependent signaling. In COS-7 cells stimulated with PRL, STAT5
appears to synergize with GC on the ß-casein promoter (25, 49, 50)
but to antagonize GC-induced MMTV-LTR promoter activity (25, 51).
Moreover, in the rat hepatoma cell line H4IIE and in COS-7 cells
stimulated with IL-6, STAT3 was shown to synergize with GC on the rat
-fibrinogen promoter and on the MMTV-LTR promoter (52). Noticeably,
many of these studies have been performed with overexpressed factors
(STAT or GR) in cells that do not normally express either STAT or GR
proteins. Our experiments showed that, in CTLL-2 cells expressing
endogenous levels of STAT5 and GR, STAT5B is necessary for IL-2
inhibition of MMTV promoter activity. Moreover, tyrosine
phosphorylation of STAT5B seems to be an important step in this
mechanism. A physical association between GR and STAT5 was present only
if the GR was activated but independently of STAT5 activation.
Formation of complexes between STAT5 and GR has previously been
described in both COS-7 cells and in HC11 mammary cells, and this
association is dependent upon ligand-induced activation of STAT5 in COS
cells but not in HC11 cells (25, 53).
In light of these observations, STAT5 appears to play a major role in
the IL-2 inhibitory effect, without binding on a specific DNA sequence
located on the MMTV promoter. Our results with the deletion mutants of
the promoter argue that integrity of sequences from -104 to +1 is
required for IL-2 inhibition. However, the MMTV-LTR proximal promoter
does not contain STAT5-specific DNA-binding sequences, suggesting that
STAT5-mediated inhibition could occur through interference with
specific factors loading on the MMTV promoter rather than through
binding to a specific DNA sequence, probably by protein-protein
interaction. Indeed, we detected a physical association between GR and
STAT5 in CTLL-2 cells. GR/STAT5 complex formation could interfere with
contacts between the GR and coactivators of the basal transcription
machinery. It has been shown in COS cells that overexpression of
CBP/p300 did not alter STAT5 inhibition of MMTV-LTR activity, despite
interaction of p300 with STAT5 and p300-dependent enhancement of
MMTV-LTR activity (51). In CTLL-2 cells, we have also found that
overexpression of CBP or SRC-1a does not alter IL-2 inhibitory effect.
Although these results rule out a mechanism of squelching, where GR and
STAT5 could compete for a limiting amount of this coactivator, they do
not exclude the possibility that another mechanism involving CBP/p300
could take place. The tyrosine phosphorylation of STAT5 appears as an
essential step for IL-2 inhibition, either by altering the conformation
of the GR-STAT5 complex, or by allowing the recruitment of other
partners. GR and STAT5 could both interact with a cofactor like
CBP/p300, and/or SRC-1a, and form a complex devoid of transcriptional
activity, as recently hypothesized for the mutual antagonism between GR
and NF-
B (54).
All together, these results indicate that in lymphocytes expressing
endogenous levels of STAT5 and GR, STAT5 plays a critical role in IL-2
regulation of GC-dependent transactivation. This could be of importance
in elucidating how cytokines modulate expression of GC-regulated genes
in pathological situations, i.e. asthma or lymphoma, or in
physiological situations, i.e. apoptosis (55).
 |
MATERIALS AND METHODS
|
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Chemicals and Reagents
Recombinant human IL-2 (Proleukin) was a gift from Chiron Corp. ( Amsterdam, The Netherlands). DEX, tetracycline, and PMA
were purchased from Sigma (LIsle DAbeau, France), and
G418 and Hygromycin B were from Life Technologies, Inc.
(Cergy-Pontoise, France). The luciferase assay system was purchased
from Promega Corp. (Madison, WI).
Plasmids
pLTR-luc plasmid contains the entire MMTV-LTR
GC-responsive promoter from the C3H strain coupled to the
luciferase reporter gene (43). p325-luc, p223-luc, p200-luc, and
p104-luc plasmids display sequential 5'-end deletions up to positions
-325, -223, -200, and -104, respectively (Fig. 5A
).
p-1,180/-860-luc plasmid displays an internal deletion between
positions -860 and -220 (43). p200-luc was obtained by deletion of
the HindIII/AflII fragment from the pLTR-luc
plasmid, filling with Klenow, and ligation. p2GRE-104-luc plasmid was
constructed by inserting two synthetic consensus GREs immediately
upstream of the -104 promoter. Briefly, the
HindIII/SacI fragment of the pLTR-luc plasmid was
ligated to the HindIII/SacI double-stranded
5'-phosphorylated oligonucleotide
(5'-AGCTTTGTACAGGATGTTCTAGATCTTGTACAGGATGTTCTGAGCT-3'). These
constructs were then verified by sequencing. The
GRE5-EBV-TATA-CAT plasmid was a kind gift of S.
Mader. The 5XTRE-tk-CAT contains five TRE sequences upstream from the
CAT (chloramphenicol acetyltransferase) gene and was a kind gift of B.
Binetruy.
The NdeI/HindIII fragment of STAT 5B was
amplified by PCR by using RSV-STAT5B (a kind gift of F. Gouilleux) as
template and oligonucleotides 5BSTART
(5'-GGGAATTCCATATGGCTATGTGGATACAGGCTCAG-3') and 5BSTOP
(5'-CCCAAGCTTGAATTCTCATGACTGTGCGTGAGGGAT-3')
as primers. The NdeI/HindIII fragment of STAT5B
was inserted at the NdeI/HindIII sites of
pGEM-Myc. The EcoRI/EcoRI fragment of Myc-STAT5B
was subsequently inserted into the PUHD 103 plasmid (33) at
the EcoRI site in the sense orientation, generating the
construct pTRE-Myc-STAT5B.
A point mutation in the STAT5B sequence from tyrosine to phenylalanine
(Y699F) was introduced using the Quick-change site-directed mutagenesis
kit (Stratagene, Amsterdam, The Netherlands). The
pTRE-Myc-STAT5B construct was used as a template and the complementary
oligonucleotides STAT5B Y699F sense (5'-TGACGGATTCGTGAAGCCACAGAT-3')
and STAT5B Y699F antisense (5'-ATCTGTGGCTTCACGAATCCGTCA-3') as primers.
The PCR was run under conditions recommended by the manufacturer for 12
cycles (30 sec at 95 C, 1 min at 55 C, and 13 min at 68 C). The
mutation in the STAT/MMTV sequence (TTCGGAGAA 224 GGCGGAGAA) was
introduced using the same kit, with the pLTR-luc and p-1,180/-860-luc
plasmids as templates and complementary oligonucleotides STAT/MMTV
sense
(5'-ACAATCTAAACAAGGCGGAGAACTCGACCTTCCTCCTG-3')
and STAT/MMTV antisense (5'-CAGGAGGAAGGTCGAGTTCTCCGCCTTGTTTAGATTGT-3')
as primers. The PCR was run for 16 cycles (30 sec at 95 C, 1 min at 55
C, and 15 min at 68 C).
The pCMV-2N3T and pCMV-2N3T-CBP plasmids were a kind gift of A.
Harel-Bellan, the pCR3.1 and
pCR3.1-SRC-1a plasmids were a kind gift of S.
Tsai.
Cell Culture, Transfections, and Tet-Off Gene Expression
System
The murine IL-2-dependent cytotoxic T cell line CTLL-2 was
cultured in complete medium: RPMI 1640 medium (Life Technologies, Inc.) containing 2 mM L-glutamine, 0.1
mg/ml streptomycin, 100 U/ml penicillin (Life Technologies, Inc.), 50 µM 2-mercaptoethanol
(Sigma), 1% sodium pyruvate (Life Technologies, Inc.), 10% FCS (Life Technologies, Inc.), and 1
ng/ml of human recombinant IL-2.
Transfections were performed using the electroporation method.
Exponentially growing CTLL-2 cells (107) were
washed in RPMI 1640 buffer and resuspended in 150 µl of RPMI 1640
containing 10 µg of plasmid. After 10 min incubation on ice, cells
were electroporated using a Bio-Rad Laboratories, Inc.
gene pulser (Ivry-sur-Seine, France) set at 250 V and 960 µF. Cells
were then maintained on ice for 10 min and resuspended in complete
medium.
For transient transfection assays with MMTV constructs, cells were
cultured with 1 ng/ml IL-2 for 48 h before transfection. After
electroporation, cells were stimulated with DEX (100 nM),
IL-2 (1 ng/ml), IL-2 plus DEX, or left untreated. After 12 h of
incubation, proteins were extracted and assayed for luciferase
activity.
Selection of stably transfected cells was initiated 48 h after
electroporation using 800 µg/ml G418 (Life Technologies, Inc.) for the cotransfections with the pMC1 plasmid (conferring
resistance to neomycin) or 800 µg/ml hygromycin B (Life Technologies, Inc.) for the cotransfections with the
pTK-hygromycin plasmid encoding an hygromycin resistance gene
(CLONTECH Laboratories, Inc. Palo Alto, CA). Stably
transfected CTLL-2 pLTR cells were selected by a 2-week treatment with
G418 and cloned by limiting dilution. Clones were then screened for
their GC-stimulated luciferase activity.
Development of a stable tTA (tetracycline transactivator) cell line was
initiated by cotransfection of CTLL-2 cells with the plasmid pUHD 151
(33) and with pMC1, encoding a neomycin resistance gene. Stably
transfected cells were selected in the presence of 800 µg/ml G418 for
2 weeks, after which CTLL-2 Tet-Off cells stably expressing the tTA
were transfected with the plasmids pTRE-Myc-STAT5B or pTRE-Myc-STAT5B
Y699F, and with pTK-hygromycin (CLONTECH Laboratories, Inc.). Cells were cultured in the presence of 1 µg/ml
tetracycline and 800 µg/ml hygromycin. Expression of the fusion
proteins Myc-STAT5B or Myc-STAT5B Y699F was turned on by removal of
tetracycline for 48 h, and analyzed by Western blotting using the
9E10 anti-Myc antibody.
Reporter Gene Activity Assays
Luciferase Assay.
Luciferase levels were measured according to the manufacturers
protocol (Promega Corp.). Briefly, extracts were prepared
by three cycles of freezing and thawing of cells resuspended in a lysis
buffer containing 25 mM Tris-phosphate, pH 7.8, 2
mM CDTA, 2 mM dithiothreitol (DTT), and
10% glycerol. Protein extracts in equivalent protein concentration
samples were mixed with 100 µl of luciferase assay reagent
(Promega Corp.). Luciferase activity was determined at 25
C after 1 min with a luminometer (LKB Wallac, Inc. Turku, Finland). Results are expressed in relative
luciferase units (RLU) relative to the basal level to which the value 1
was arbitrarily affected. Fold induction was calculated as the ratio of
arbitrary luciferase units in cells treated with IL-2, DEX, or DEX and
IL-2 compared with untreated cells. Fold induction (DEX-treated
cells) = (DEX activity - basal activity)/basal activity.
Fold induction (DEX + IL-2-treated cells) = (DEX + IL-2 activity
- IL-2 activity)/basal activity.
CAT Assay.
Extracts were prepared by three cycles of freezing and thawing of cells
resuspended in hypotonic buffer (0.25 M Tris HCl, pH 8).
Protein extracts (40 µg) were incubated with
(14C)-chloramphenicol (60 mCi/mmol,
Amersham Pharmacia Biotech, Orsay, France) in the presence
of 2 mM acetyl coenzyme A (Sigma) for 1 h
at 37 C. Acetylated chloramphenicol was extracted in ethyl acetate and
separated from unmodified chloramphenicol by TLC. Conversion of
chloramphenicol was quantified using a Storm 840 phosphorimager and
the Imagequant software (Molecular Dynamics, Inc.,
Sunnyvale, CA). Percentage of chloramphenicol conversion represents the
ratio between acetylated chloramphenicol and total chloramphenicol
(acetylated and nonacetylated).
Preparation of Nuclear Extracts
Nuclear extracts were prepared by a modification of the method
described by Dignam et al. (56). Cells were deprived of IL-2
for 3 h and stimulated at 37 C for 1 h with DEX, IL-2, or DEX
plus IL-2. Cells were then pelleted, washed with ice-cold 1x PBS, and
maintained for 10 min on ice in a hypotonic buffer containing 10
mM HEPES, pH 7.8, 15 mM
KCl, 2 mM MgCl2, 1
mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 1 mM DTT.
Cytoplasmic membranes were lysed by 50 strokes using a Kontes all-glass
Dounce homogenizer (B type pestle). The lysate was centrifuged at
1,000 x g for 5 min at 4 C, and the nuclear pellet was
resuspended in a high-salt buffer (20 mM HEPES,
pH 7.8, 1.5 mM MgCl2, 0.2
mM EDTA, 25% (vol/vol) glycerol, 1
mM phenylmethylsulfonyl fluoride (PMSF), 1
µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1
mM DTT, 400 mM NaCl).
Nuclear extracts were centrifuged at 15,000 x g for 20
min at 4 C.
EMSA
Oligonucleotides were purchased from Oligo Express (Paris,
France). Complementary sequences were annealed at 80 C for 10 min and
65 C for 10 min and then were end-labeled using
(32P)-ATP with T4 polynucleotide kinase
(Life Technologies, Inc.) and used for EMSA after ethanol
precipitation.
The 5'-TCTTTTGGAATTTATCCAAATCTTAT-3' probe was used for
NF-1 binding. The
5'-ATCTTATGTAAATGCTTATGTAAACCAAGA- 3' probe was
used for Oct binding. These probes correspond, respectively, to the
NF-1 and Oct binding sites found in the MMTV promoter between positions
-80 and -55 for NF-1, -61 and -32 for Oct-1. The GAS probe from the
GAS site of the Fc
R promoter
(5'-GTATTTCCCAGAAAAGGAAC-3') was used for STAT5 binding,
and the STAT/MMTV probe
(5'-ATCTAAACAATTCGGAGAACTCGACCTTC-3') corresponds to the
STAT response element located between positions -923 and -914 of the
MMTV promoter. Specificity was determined by using a 25-fold molar
excess of cold probe or random probe
(5'-CCTCCATGACTCCAGAACTAACCTCCATGAC-3').
End-labeled oligonucleotides were incubated at 25 C for 30 min with 15
µg of nuclear proteins in the presence of 1 µg of sonicated salmon
sperm DNA in 20 µl of binding buffer (12% glycerol, 12
mM HEPES, pH 7.8, 60 mM KCl, 1 mM
EDTA, and 1 mM DTT). Protein-DNA complexes were separated
from free probe on a 5% polyacrylamide gel in 0.5x TBE running buffer
at 200 V. For supershift experiments, 3 µg of the control IgG or of
the specific anti-Oct-1 IgG (C-21, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or 1.5 µl of the anti-STAT5A and
anti-STAT5B sera were preincubated with nuclear extracts for 2 h
at 4 C before addition of the probe.
Production and DNA Binding Activity of Recombinant GR
The recombinant rat GR was produced using the TNT Quick coupled
transcription/translation system as recommended by the manufacturer
(Promega Corp.). Briefly, 1 µg of pET30rGR (containing
the rat GR gene under the control of the T7 promoter) was added to an
aliquot of TNT Quick master mix and incubated in a 50 µl reaction
volume for 90 min at 30 C. The synthesized GR was then activated for
1 h at 4 C and 1 additional hour at 30 C in a buffer containing 10
mM HEPES, pH 7.4, 20 mM ß-mercaptoethanol,
5% glycerol, 50 mM NaCl, and 1 µM DEX. The
5'-ATCTCTGCAGAACAGGATGTTCTAGCTACTT-3' probe was
used for GR DNA binding. Specificity was determined by using a 50-fold
molar excess of cold probe or random probe. End-labeled GRE
oligonucleotides were incubated at 25 C for 1 h with 2 µl of
activated lysate in the presence of 1 µg of sonicated salmon sperm
DNA in 20 µl of binding buffer (12% glycerol, 12 mM
HEPES, pH 7.8, 60 mM KCl, 1 mM EDTA, and 1
mM DTT). For competition experiments, 1 µg, 2.5 µg, or
5 µg of nuclear extracts from CTLL-2 cells deprived of IL-2 for
3 h and treated or not with IL-2 for 1 h were incubated with
the activated lysate. For supershift experiments, 1 µg of the control
IgG or of the specific anti-GR (M-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated with GR for 30 min at 4
C before addition of the probe.
DNA Affinity Precipitation of STAT Proteins
Cells were deprived of IL-2 for 2 h and treated with IL-2
(500 pg/ml) for 12 h at 37 C. Cells were then collected by
centrifugation, washed in 1x PBS, and resuspended in NP40 buffer (50
mM Tris HCl, pH 8, 0.5% NP40, 150 mM NaCl, 0.1
mM EDTA, 10 mM NaF, 1 mM PMSF, 1
µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM DTT). The
double-stranded 5'-biotinylated oligonucleotide GAS was coupled to
streptavidin-agarose beads (Sigma) for 1 h at 4 C.
Whole-cell extracts were then incubated with the precoated beads for
1 h at 4 C. The beads were then washed three times with the NP40
lysis buffer and boiled in reducing sample buffer to elute the
complexes. Bound proteins were then separated on 8% polyacrylamide gel
and electroblotted onto Amersham Pharmacia Biotech PVDF
(polyvinylidene difluoride) membranes. Western blot
analysis was performed with the specific anti-STAT5B antibody
(ref 06969, Upstate Biotechnology, Inc. Lake Placid,
NY).
Coimmunoprecipitation Assays
CTLL-2 cells were deprived of IL-2 for 3 h and then
stimulated for 1 h with IL-2 (1 ng/ml) and/or DEX (100
nM), or left untreated. Cell lysates were first incubated
for 30 min at 4 C with protein A sepharose beads (Sigma)
and preimmune serum and then centrifuged at 4,000 rpm for 1 min. The
supernatant (precleared lysate) was incubated overnight at 4 C with the
anti-GR antibody (BuGR2, Affinity BioReagents, Inc., Golden, CO) precoupled to Protein A sepharose beads. Immune
complexes were washed three times with lysis buffer and analyzed by
SDS-PAGE using anti-STAT5 antiserum.
Western Blot
Cells were collected by centrifugation, washed in 1xPBS, and
resuspended in NP40 buffer (50 mM Tris HCl, pH 8, 0.5%
NP40, 150 mM NaCl, 0.1 mM EDTA, 10
mM NaF, 1 mM PMSF, 1 µg/ml aprotinin, 1
µg/ml leupeptin, 1 mM DTT). Cell lysates were resolved by
SDS-PAGE on 8% polyacrylamide gels and electroblotted onto
Amersham Pharmacia Biotech PVDF membranes. After
saturation of nonspecific binding sites with dry low-fat milk in
TBS-Tween 20 (0.2%) for 2 h, membranes were probed with the
monoclonal 9E10 anti-Myc monoclonal antibody and developed with ECL
(Amersham Pharmacia Biotech).
 |
ACKNOWLEDGMENTS
|
---|
The authors specially acknowledge Karine Andréau and
Marie-Liesse Asselin for helpful discussion and participation in some
of the experiments. The authors thank Michel Renoir, Véronique
Marsaud, and José Luis-Zugaza for critical reading of this
manuscript. The authors thank Fabrice Gouilleux for the kind gift of
RSV-STAT5B plasmid and anti-STAT5A and STAT5B sera, Sylvie Mader for
the pGRE5-EBV-TATA-CAT plasmid, Bernard Binetruy for the 5XTRE-tk-CAT
plasmid, Annick Harel-Bellan for the pCMV-2N3T and pCMV-2N3T-CBP
plasmids, Sophia Tsai for the pCR3.1 and
pCR3.1-SRC-1a plasmids, and Josiane Pierre for
making available the tet-system components (pUHD 151, pUHD 103, and
pUHC 133 plasmids) of Hermann Bujard (Zentrum für Molekulare
Biologie der Universität Heidelberg). We thank Sophie Amsellem
for raising the anti-STAT5 antibody and gratefully acknowledge the
technical assistance of Sophie Gruel and Chantal Broch.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Marc Pallardy, INSERM U461, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry cedex, France. E-mail: marc.pallardy{at}cep.u-psud.fr
This research was supported by INSERM and by a fellowship from the
Association pour la Recherche sur le Cancer to Armelle Biola.
1 Present address: INSERM, 101 rue de Tolbiac, 75654 Paris,
France. 
Received for publication June 9, 2000.
Revision received March 7, 2001.
Accepted for publication March 12, 2001.
 |
REFERENCES
|
---|
-
Watson CE, Archer TK 1998 Chromatin and
steroid-receptor-mediated transcription. In: Freedman LP (ed)
Molecular Biology of Steroid and Nuclear Hormone Receptors.
Birkhäuser, Boston, pp 209235
-
Bagchi MK 1998 Molecular mechanisms of nuclear
receptor-mediated transcriptional activation and basal repression. In:
Freedman LP (ed) Molecular Biology of Steroid and Nuclear Hormone
Receptors. Birkhäuser, Boston, pp 159189
-
Vottero A, Chrousos 1999 Glucocorticoid receptor ß:
view I. Trends Endocrinol Metab 10:333338[CrossRef][Medline]
-
Carlstedt-Duke J 1999 Glucocorticoid receptor ß: view
II. Trends Endocrinol Metab 10:339342[CrossRef][Medline]
-
Chandler VL, Maler BA, Yamamoto KR 1983 DNA sequences bound
specifically by glucocorticoid receptor in vitro render a heterologous
promoter hormone responsive in vivo. Cell 33:489499[Medline]
-
Scheidereit C, Geisse S, Westphat HM, Beato M 1983 The
glucocorticoid receptor binds to defined nucleotide sequences near the
promoter of the mouse mammary tumour virus. Nature 304:749752[Medline]
-
Buetti E, Kühnel B, Diggelmann 1989 Dual function of a
nuclear factor I binding site in MMTV transcription regulation. Nucleic
Acids Res 17:30653078[Abstract]
-
Buetti E 1994 Stably integrated mouse mammary tumor virus
long terminal repeat DNA requires the octamer motifs for basal promoter
activity. Mol Cell Biol 14:11911203[Abstract]
-
Préfontaine GG, Walther R, Giffin W, Lemieux ME, Pope
L, Haché RJG 1999 Selective binding of steroid hormone receptors
to octamer transcription factors determines transcriptional synergism
at the mouse mammary tumor virus promoter. J Biol Chem 274:2671326719[Abstract/Free Full Text]
-
Cavin C, Buetti E 1995 Tissue-specific and ubiquitous factors
binding next to the glucocorticoid receptor modulate transcription from
the mouse mammary tumor virus promoter. J Virol 69:37593770[Abstract]
-
Archer TK, Lefebvre P, Wolford RG, Hager GL 1992 Transcription
factor loading on the MMTV promoter: a bimodal mechanism for promoter
activation. Science 255:15731576[Medline]
-
Bertoglio JH, Leroux E 1988 Differential effect of
glucocorticoids on the proliferation of a murine helper and a cytolytic
T cell clone in response to IL-2 and IL-4. J Immunol 141:11911196[Abstract/Free Full Text]
-
Louahed J, Renauld JC, Demoulin JB, Baughman G, Bourgeois S,
Sugamura K, Van Snick J 1996 Differential activity of dexamethasone on
IL-2-, IL-4-, or IL-9-induced proliferation of murine
factor-dependent T cell lines. J Immunol 156:37043710[Abstract]
-
Ramirez F 1998 Glucocorticoids induce a Th2 response in vitro.
Dev Immunol 6:233243[Medline]
-
Fernandez-Ruiz E, Rebollo A, Nieto MA, Sanz E, Somoza C,
Ramirez F, Lopez-Rivas A, Silva A 1989 IL-2 protects T cell hybrids
from the cytolytic effect of glucocorticoids. Synergistic effect of
IL-2 and dexamethasone in the induction of high-affinity IL-2
receptors. J Immunol 143:41464151[Abstract/Free Full Text]
-
Nieto MA, Lopez-Rivas A 1989 IL-2 protects T lymphocytes from
glucocorticoid-induced DNA fragmentation and cell death. J Immunol 143:41664170[Abstract/Free Full Text]
-
Mor F, Cohen IR 1996 IL-2 rescues antigen-specific T cells
from radiation or dexamethasone-induced apoptosis. J Immunol 156:515522[Abstract]
-
Perrin-Wolff M, Mishal Z, Bertoglio J, Pallardy M 1996 Position 16 of the steroid nucleus modulates glucocorticoid-induced
apoptosis at the transcriptional level in murine T-lymphocytes. Biochem
Pharmacol 52:14691476[CrossRef][Medline]
-
Zubiaga AM, Munoz E, Huber BT 1992 IL-4 and IL-2 selectively
rescue Th cell subsets from glucocorticoid-induced apoptosis. J
Immunol 149:107112[Abstract/Free Full Text]
-
Renaud JC, Vink A, Louahed J, Van Snick J 1995 Interleukin-9
is a major anti-apoptotic factor for thymic lymphoma. Blood 85:13001305[Abstract/Free Full Text]
-
Herrlich P, Göttlicher M 1998 Transcriptional cross talk
by steroid hormone receptors. In: Freedman LP (ed) Molecular Biology of
Steroid and Nuclear Hormone Receptors. Birkhäuser, Boston, pp
191207
-
Maroder M, Farina AR, Vacca A, Felli MP, Meco D, Screpanti I,
Frati L, Gulino A 1993 Cell-specific bifunctional role of Jun oncogene
family members on glucocorticoid receptor-dependent transcription. Mol
Endocrinol 7:570584[Abstract]
-
Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M 1995 Immunosuppression by glucocorticoids: inhibition of NF
B
activity through induction of I
B synthesis. Science 270:286290[Abstract]
-
Scheinman RI, Cogswell PC, Lofquist AK, Baldwin Jr AS 1995 Role of transcriptional activation of I
B
in mediation of
immunosuppression by glucocorticoids. Science 270:283286[Abstract]
-
Stöcklin E, Wissler M, Gouilleux F, Groner B 1996 Functional interactions between Stat5 and the glucocorticoid receptor.
Nature 383:726728[CrossRef][Medline]
-
Gaffen SL, Lai SY, Gouilleux F, Groner B, Goldsmith MA, Greene
WC 1995 Signaling through the interleukin-2 receptor ß chain
activates a STAT-5-like DNA-binding activity. Proc Natl Acad Sci USA 92:7192196[Abstract]
-
Wakao H, Harada N, Kitamura T, Mui A, Miyajima A 1995 Interleukin-2 and erythropoietin activate STAT5/MGF via distinct
pathways. EMBO J 14:25272535[Abstract]
-
Kim MH, Peterson DO 1995 Oct-1 protein promotes functional
transcription complex assembly on the mouse mammary tumor virus
promoter. J Biol Chem 270:2782327828[Abstract/Free Full Text]
-
Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H,
Herrlich P 1990 Antitumor promotion and antiinflammation:
down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone.
Cell 62:11891204[Medline]
-
Schüle R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J,
Yang N, Verma IM, Evans RM 1990 Functional antagonism between
oncoprotein c-Jun and the glucocorticoid receptor. Cell 62:12171226[Medline]
-
Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ,
Drouin J, Karin M 1990 Transcriptional interference between c-Jun and
the glucocorticoid receptor: mutual inhibition of DNA binding due to
direct protein-protein interaction. Cell 62:12051215[Medline]
-
Walker PR, Kwast-Welfeld J, Gourdeau H, Leblanc J, Neugebauer
W, Sikorska M 1993 Relationship between apoptosis and the cell cycle in
lymphocytes: roles of protein kinase C, tyrosine phosphorylation, and
AP-1. Exp Cell Res 207:142151[CrossRef][Medline]
-
Gossen M, Bujard H 1992 Tight control of gene expression in
mammalian cells by tetracycline-responsive promoters. Proc Natl Acad
Sci USA 89:55475551[Abstract]
-
Kam JC, Szefler SJ, Surs W, Sher ER, Leung DYM 1993 Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding
affinity and T cell response to glucocorticoids. J Immunol 151:34603466[Abstract/Free Full Text]
-
Riccardi C, Cifone MG, Migliorati G 1999 Glucocorticoid
hormone-induced modulation of gene expression and regulation of T-cell
death: role of GITR and GILZ, two dexamethasone-induced genes. Cell
Death Differ 6:11821189[CrossRef][Medline]
-
Karnitz LM, Abraham RT 1996 Interleukin-2 receptor signaling
mechanisms. Adv Immunol 61:147199[Medline]
-
Gesbert F, Delespine-Carmagnat M, Bertoglio J 1998 Recent
advances in the understanding of interleukin-2 signal transduction.
J Clin Immunol 18:307320[CrossRef][Medline]
-
White JH, McCuaig KA, Mader S 1994 A simple and sensitive
high-throughput assay for steroid agonists and antagonists.
Biotechnology 12:10031007[Medline]
-
Kalff M, Gross B, Beato M 1990 Progesterone receptor
stimulates transcription of mouse mammary tumour virus in a cell-free
system. Nature 344:360362[CrossRef][Medline]
-
Préfontaine GG, Lemieux ME, Giffin W, Schild-Poulter C,
Pope L, LaCasse E, Walker P, Haché JG 1998 Recruitment of octamer
transcription factors to DNA by glucocorticoid receptor. Mol Cell Biol 18:34163430[Abstract/Free Full Text]
-
Günzburg WH, Salmons B 1992 Factors controlling the
expression of mouse mammary tumour virus. Biochem J 283:625632[Medline]
-
Le Ricousse S, Gouilleux F, Fortin D, Joulin V, Richard-Foy H 1996 Glucocorticoid and progestin receptors are differently involved in
the cooperation with a structural element of the mouse mammary tumor
virus promoter. Proc Natl Acad Sci USA 93:50725077[Abstract/Free Full Text]
-
Lefebvre P, Berard DS, Cordingley MG, Hager GL 1991 Two
regions of the mouse mammary tumor virus long terminal repeat regulate
the activity of its promoter in mammary cell lines. Mol Cell Biol 11:25292537[Medline]
-
Gouilleux F, Sola B, Couette B, Richard-Foy H 1991 Cooperation
between structural elements in hormone-regulated transcription from the
mouse mammary tumor virus promoter. Nucleic Acids Res 19:15631569[Abstract]
-
Siddique HR, Sarkar NH 1990 The interactions of a c-Jun/Fos
related protein factor with the U3 sequences of the mouse mammary tumor
virus LTR. Biochem Biophys Res Commun 172:348356[Medline]
-
Tanaka H, Dong Y, McGuire J, Okret S, Poellinger L, Makino I,
Gustafsson JA 1993 The glucocorticoid receptor and a putative repressor
protein coordinately modulate glucocorticoid responsiveness of the
mouse mammary tumor virus promoter in the rat hepatoma cell line M1.19.
J Biol Chem 268:18541859[Abstract/Free Full Text]
-
Guizani L, Perrin-Wolff M, Breard J, Binetruy B, Bertoglio J 1996 Mechanisms in interleukin-2 protection against
glucocorticoid-induced apoptosis: regulation of AP-1 and glucocorticoid
receptor transcriptional activities. J Interferon Cytokine Res 16:601609[Medline]
-
Shemshedini L, Knauthe R, Sassone-Corsi P, Pornon A,
Gronemeyer H 1991 Cell-specific inhibitory and stimulatory effects of
Fos and Jun on transcription activation by nuclear receptor. EMBO J 10:38393849[Abstract]
-
Stoecklin E, Wissler M, Moriggl R, Groner B 1997 Specific DNA
binding of STAT5, but not of glucocorticoid receptor, is required for
their functional cooperation in the regulation of gene transcription.
Mol Cell Biol 17:67086716[Abstract]
-
Lechner J, Welte T, Tomasi JK, Bruno P, Cairns C, Gustafson
J-A, Doppler W 1997 Promoter-dependent synergy between glucocorticoid
receptor and STAT5 in the activation of ß-casein gene
transcription. J Biol Chem 272:2095420960[Abstract/Free Full Text]
-
Pfitzner E, Jähne R, Wissler M, Stoeklin E, Groner B 1998 p300/CREB-binding protein enhances the prolactin-mediated
transcriptional induction through direct interaction with the
transactivation domain of Stat5, but does not participate in the
Stat5-mediated suppression of the glucocorticoid response. Mol
Endocrinol 12:15821593[Abstract/Free Full Text]
-
Zhang Z, Jones S, Hagood JS, Fuentes NL, Fuller G 1997 STAT3
as a co-activator of glucocorticoid receptor. J Biol Chem 272:3060730610[Abstract/Free Full Text]
-
Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a
and Stat5b homodimers and heterodimers and their association with the
glucocorticoid receptor in mammary cells. Mol Cell Biol 18:17831792[Abstract/Free Full Text]
-
McKay LI, Cidlowski JA 2000 CBP (CREB binding protein)
integrates NF-
B (nuclear factor-
B) and glucocorticoid receptor
physical interactions and antagonism. Mol Endocrinol 14:12221234[Abstract/Free Full Text]
-
Demoulin J-B, Van Roost E, Stevens M, Groner B, Renauld
J-C 1999 Distinct roles for STAT1, STAT3, and STAT in differentiation
gene induction and apoptosis inhibition by interleukin-9. J Biol
Chem 274:2585525861[Abstract/Free Full Text]
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]