Stat5b Inhibits NF
B-Mediated Signaling
Guoyang Luo and
Li-yuan Yu-Lee
Department of Microbiology and Immunology (G.L., L.-y.Y.-L.),
Medicine (L.-y.Y.-L.), and Molecular and Cellular Biology
(L.-y.Y.-L.) Baylor College of Medicine Houston, Texas
77030
 |
ABSTRACT
|
---|
Signal transducers and activators of
transcription (Stat) are latent transcription factors that participate
in cytokine signaling by regulating the expression of early response
genes. Our previous studies showed that Stat5 functions not only as a
transcriptional activator but also as a transcriptional inhibitor,
depending on the target promoter. This report further investigates the
mechanism of Stat5b-mediated inhibition and demonstrates that
PRL-inducible Stat5b inhibits nuclear factor
B (NF
B) signaling to
both the interferon regulatory factor-1 promoter and to the
thymidine kinase promoter containing multimerized NF
B elements
(NF
B-TK). Further, PRL-inducible Stat5b inhibits tumor necrosis
factor-
signaling presumably by inhibiting endogenous NF
B. This
Stat5b-mediated inhibitory effect on NF
B signaling is independent of
Stat5b-DNA interactions but requires the carboxyl terminus of Stat5b as
well as Stat5b nuclear translocation and/or accumulation, suggesting
that Stat5b is competing for a nuclear factor(s) necessary for
NF
B-mediated activation of target promoters. Increasing
concentrations of the coactivator p300/CBP reverses Stat5b inhibition
at both the interferon-regulatory factor-1 and NF
B-TK promoters,
suggesting that Stat5b may be squelching limiting coactivators via
protein-protein interactions as one mechanism of promoter inhibition.
These results further substantiate our observation that Stat factors
can function as transcriptional inhibitors. Our studies reveal
cross-talk between the Stat5b and NF
B signal transduction pathways
and suggest that Stat5b-mediated inhibition of target promoters
occurs at the level of protein-protein interactions and involves
competition for limiting coactivators.
 |
INTRODUCTION
|
---|
Cellular responses to activation by multiple cytokines depend on
the expression of specific cytokine receptors, the complement of
signaling molecules, and the stage of differentiation of the responding
cells. Cytokines elicit numerous biological responses by activating a
variety of latent transcription factors, including signal transducers
and activators of transcription (Stats) and nuclear factor of kappa B
(NF
B). Although their activation pathways are different, activated
Stats and NF
B translocate into the nucleus and function either
individually or cooperatively in regulating the expression of target
genes (1 2 3 ). Interactions among various cytoplasmic as well as nuclear
factors, the promoter context of the target gene, and the presence of
coactivator complexes determine the expression of the target gene and
hence the biological outcome of cytokine actions.
PRL, a pituitary peptide hormone as well as a T cell cytokine, plays a
modulatory role in various aspects of the immune response (4 ). The
immunomodulatory effect is mediated by the binding of PRL to the
PRL-receptor (PRL-R), which is a member of the hematopoietin/cytokine
receptor superfamily. The interaction of PRL and PRL-R leads to the
activation of the protein tyrosine kinase JAK2 and a number of Stat
factors (5 ). Stat1, Stat3, Stat5a, and Stat5b have been shown to be
rapidly tyrosine phosphorylated in response to PRL stimulation (6 7 8 ).
Activated Stats translocate into the nucleus and bind to the interferon
activation sequence (GAS) in the promoter region of target genes
and regulate the transcription of these genes. Our previous studies
have shown that PRL stimulates the transcription of the immediate early
gene interferon regulatory factor 1 (IRF-1) in Nb2 T cells (6 9 ).
Promoter analysis has shown that the -200 bp promoter proximal region
is responsible for mediating PRL induction of the IRF-1 gene (6 ).
Further, a GAS element at -112 bp has been shown to function as a
PRL-responsive enhancer element in the IRF-1 promoter. Stat1 has been
shown to bind to the IRF-1 GAS in a PRL-inducible manner and positively
mediates PRL activation of the IRF-1 promoter (10 ). Stat5 has also been
demonstrated to bind the IRF-1 GAS in a PRL-inducible manner (11 ).
Unexpectedly, Stat5 inhibits PRL induction of the IRF-1 promoter. The
inhibitory effect does not require Stat5 to bind DNA, suggesting that
the inhibition may be mediated via protein/protein interaction by
competing for factor(s) that is necessary for PRL induction of the
IRF-1 promoter (11 12 ).
In addition to the GAS element, a number of other recognition sites for
DNA-binding proteins, such as NF
B, are present in the IRF-1 promoter
(13 ). Recent studies have shown that IFN
-activated Stat1 and tumor
necrosis factor-
(TNF
)-activated NF
B function synergistically
at the IRF-1 promoter to induce IRF-1 expression (1 2 ). These studies
suggest that IRF-1 gene expression can be cooperatively regulated by
both the JAK/Stat signaling pathway and NF
B signaling pathway. In
this report, we examined the negative cross-talk between Stat5b and
NF
B signaling at the IRF-1 promoter. We show that Stat5b inhibits
p50/p65 NF
B signaling to the IRF-1 promoter as well as to the
heterologous thymidine kinase (TK) promoter containing multimerized
NF
B elements. Stat5b-mediated inhibitory action requires the
presence of the carboxyl terminus and the nuclear translocation of
Stat5b, but does not require Stat5b to bind to the GAS element. Stat5b
inhibition appears to be mediated by protein/protein interactions, in
particular, by squelching of limiting amounts of the p300/CBP
coactivators, as one mechanism of Stat5b transcriptional inhibition at
target promoters. These results suggest that Stat5b inhibits NF
B
signaling by competing for coactivators necessary for NF
B-mediated
gene transcription.
 |
RESULTS
|
---|
Stat5b Inhibits NF
B Induction of the IRF-1 Promoter
Our previous studies have shown that Stat5b inhibits PRL
induction of the IRF-1 promoter in transient transfection assays (11 ).
This inhibitory effect does not require Stat5b to bind to DNA,
suggesting that Stat5b competes for factor(s) necessary for PRL
induction of the IRF-1 promoter (11 ). To determine what factor(s)
Stat5b may be competing for, we first examined other transcription
factors that have potential binding sites at the IRF-1 promoter. One
such factor is NF
B, which has a binding site (-35 to -45 bp) 3' to
the PRL-responsive GAS element (-112 bp) in the IRF-1 promoter (Fig. 1
). This NF
B element has been shown to
mediate TNF
induction of the IRF-1 promoter in HepG2 and NIH3T3
cells (1 2 ). The TNF
-mediated induction of the IRF-1 promoter is
further synergized by INF
via activation of Stat1 binding to the GAS
element. To determine whether Stat5b may affect NF
B signaling to the
IRF-1 promoter, COS cells were cotransfected with the PRL-R, Stat5b,
p50 NF
B, p65 NF
B, and the 1.7-kb IRF-1-chloramphenicol
acetyltransferase (CAT) constructs. In this reconstituted COS cell
transfection system, PRL stimulates a 2- to 3-fold induction of the
IRF-1 promoter, which is inhibited by Stat5b (Fig. 1
) as shown
previously (11 ). Cotransfection of p50/65 NF
B led to a 5- to 6-fold
induction of the IRF-1 promoter in the absence of PRL stimulation (Fig. 1
). PRL stimulation further enhanced IRF-1 promoter activity above that
induced by NF
B, presumably due to the activation of endogenous Stat1
(10 ). In the absence of the PRL stimulation, overexpression of Stat5b
did not affect NF
B-mediated induction of the IRF-1 promoter. In
contrast, upon PRL stimulation, Stat5b strongly inhibited both PRL and
NF
B-mediated IRF-1 promoter activity in a dose-dependent manner
(Fig. 1
). These studies show that NF
B can activate the IRF-1
promoter and this induction is inhibited by Stat5b in a dose-dependent
as well as PRL-dependent manner.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 1. Stat5b Inhibits NF B Signaling to the IRF-1
Promoter
COS cells were transiently cotransfected with 1 µg of the Nb2 PRL-R,
0.1 µg of the 1.7-kb IRF-1-CAT, each of p50/p65 NF B and Stat5b as
indicated, stimulated with 100 ng/ml of PRL for 24 h, and assayed
for CAT expression as described in Materials and
Methods. The data are summarized from multiple independent
experiments (n = 5).
|
|
The IRF-1 GAS Element Does Not Mediate Stat5b Inhibition of NF
B
Signaling to the IRF-1 Promoter
Next, we determined whether Stat5b inhibition of NF
B signaling
requires the presence of a functional GAS element at the IRF-1
promoter. An intact IRF-1 GAS element is critical for PRL induction of
the IRF-1 promoter (Fig. 2
), as
site-directed mutations in the GAS element abolished PRL activation, as
was shown previously (10 ). However, mutation of this GAS element did
not affect p50/p65 NF
B-mediated activation of the IRF-1 promoter,
confirming that NF
B activates the IRF-1 promoter independently of a
functional GAS element. This NF
B-mediated signaling to the mutant
GAS IRF-1 promoter was still inhibited by Stat5b in a PRL-dependent
manner. These results clearly show that Stat5b inhibition of NF
B
signaling to the IRF-1 promoter does not require Stat5b to interact
with the GAS element.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 2. Stat5b Inhibition of NF B Signaling Is
Independent of the GAS Element
COS cells were transiently cotransfected as described in Fig. 1 , except
that the 1.7-kb mutant GAS IRF-1-CAT reporter was used (n = 3).
|
|
Stat5b Inhibits NF
B Activation of the Heterologous NF
B-TK
Promoter
To further determine whether Stat5b-mediated inhibitory effects
can be demonstrated at a promoter that only contains NF
B elements,
the heterologous TK promoter containing two copies of the NF
B
element was examined (14 ). As expected, the NF
B-TK promoter was not
responsive to PRL stimulation, as it does not have a GAS element (Fig. 3
). p50/p65 NF
B mediated greater than
10-fold induction of the NF
B-TK promoter, which again is not further
inducible by PRL stimulation. Interestingly, Stat5b inhibited
the NF
B-mediated activation of this heterologous TK promoter, in a
PRL- as well as DNA dose-dependent manner (Fig. 3
). These results
demonstrate that Stat5b can inhibit NF
B signaling to promoters that
do not contain a GAS element. These results further support our
hypothesis that Stat5b-mediated inhibition of NF
B signaling is not
mediated by Stat-DNA interactions but is mediated by protein-protein
interactions.
Stat5b-Mediated Inhibition Requires Its Nuclear
Localization
To further understand how Stat5b inhibits NF
B-mediated
signaling to both the IRF-1 and NF
B TK promoters, a Stat5b DNA
binding mutant, Stat5b VVVI (11 ), was tested for its ability to inhibit
NF
B signaling to either promoter. Previous studies have shown that
the Stat5b VVVI mutant strongly inhibits PRL signaling to the IRF-1
promoter (11 ). First, immunofluorescence microscopy was used to examine
the intracellular localization of Stat5b and Stat5b VVVI mutant after
PRL stimulation in transfected COS cells. Using deconvolution confocal
microscopy, wild-type Stat5b immunofluorescence staining
(red) was detected throughout the transfected COS cell in
the absence of PRL stimulation (Fig. 4A
).
Others have also observed the general staining of inactive Stats in the
nucleus of transfected cells (15 16 ). However, upon PRL stimulation,
Stat5b exhibited a punctate staining pattern in the nucleus, indicating
PRL-inducible nuclear translocation of Stat5b (Fig. 4B
). Similarly, in
unstimulated cells, Stat5b VVVI mutant also showed general staining
throughout the cytoplasm with a low level of staining detected in the
nucleus (Fig. 4C
). In contrast, with PRL stimulation, the Stat5b VVVI
mutant remained primarily in the cytoplasm, often with increased
staining around the perinuclear region (Fig. 4D
). Although a low level
of Stat5b VVVI staining was found in the nucleus, the general staining
pattern of the Stat5b VVVI mutant is consistently distinct from the
clear punctate nuclear staining of wild-type Stat5b in response to PRL
stimulation (compare Fig. 4D
with Fig. 4B
). These results suggest that
Stat5b VVVI mutant does not translocate into or accumulate effectively
in the nucleus upon PRL stimulation.
The defect in nuclear accumulation of Stat5b VVVI mutant is correlated
with the inability of this mutant to inhibit NF
B signaling to either
the IRF-1 (Fig. 4E
) or NF
B-TK (Fig. 4F
) promoter. Thus, wild-type
Stat5b still inhibits NF
B signaling to both the IRF-1 and NF
B TK
promoters (Fig. 4
, E and F), but the Stat5b VVVI mutant fails to
inhibit NF
B signaling to these promoters. Note that the Stat5b VVVI
mutant was still capable of inhibiting that portion of IRF-1 promoter
activity that is inducible by PRL, but not by NF
B (open and
solid bars are not significantly different in the Stat5b VVVI
transfected cells in Fig. 4E
). These results show that Stat5b-mediated
inhibition of NF
B signaling requires its nuclear translocation.
Further, these results imply that Stat5b-mediated inhibition of
PRL-inducible Stat1 signaling can occur at both the nuclear as well as
extranuclear levels.
Stat5b Inhibits TNF
Signaling
We next examined whether Stat5b can inhibit endogenous NF
B
activity as stimulated via TNF
, in the absence of p50/p65 NF
B
overexpression. COS cells were cotransfected with the PRL-R, Stat5b, or
two Stat5b mutants, Stat5b
40C or Stat5b VVVI, and either 1.7-kb
IRF-1-CAT or NF
B TK-CAT constructs. PRL or TNF
individually
stimulated 3- and 2-fold induction, respectively, of IRF-1 promoter
activity (Fig. 5A
). TNF
plus PRL
together stimulated 5- to 6-fold induction of the IRF-1 promoter in
vector-transfected control cells (Fig. 5A
), presumably due to the
activation of endogenous NF
B and endogenous Stat1, respectively. In
the presence of Stat5b, PRL stimulation resulted in a reduction in
TNF
-inducible IRF-1 promoter activity. These results show that
PRL-inducible Stat5b can inhibit TNF
-inducible NF
B signaling to
the IRF-1 promoter. Furthermore, PRL-inducible Stat5b also
inhibited the large 7- to 8-fold TNF
-inducible NF
B signaling to
the heterologous NF
B-TK promoter (Fig. 5B
).
Previous studies have shown that the Stat5b
40C mutant, which lacks
the carboxyl-terminal 40 amino acids, fails to inhibit Stat1-mediated
PRL signaling to the IRF-1 promoter (11 ). The Stat5b
40C mutant also
failed to inhibit TNF
signaling to both the IRF-1 (Fig. 5A
) and
NF
B-TK promoters (Fig. 5B
), suggesting that the carboxyl terminus of
Stat5b is involved in inhibition at these promoters. On the other hand,
the Stat5b VVVI mutant, which does not accumulate significantly
in the nucleus, clearly inhibited PRL signaling to the IRF-1
promoter (Fig. 5A
), but failed to inhibit TNF
signaling to either
the IRF-1 (Fig. 5A
) or NF
B-TK (Fig. 5B
) promoter. In agreement with
the data on overexpression of p50/p65 NF
B (Fig. 4
), these results
suggest that Stat5b inhibits TNF
signaling by inhibiting endogenous
NF
B, and that this inhibition involves the carboxyl terminus of
Stat5b and requires Stat5b to be translocated into the nucleus.
Exogenous p300/CBP Coactivators Reverse Stat5b-Mediated Inhibition
at Target Promoters
Recent studies have shown that the activities of Stat factors can
be modulated by their interactions with other DNA-binding proteins and
non-DNA-binding proteins such as coactivators. p300/CBP has been shown
to interact with Stat1 (17 18 19 ), Stat2 (20 ), and Stat5a (21 ) as well as
with NF
B (22 23 24 ) to enhance target gene expression. For example,
IFN
-inducible Stat2 appears to compete with TNF
-inducible NF
B
for the coactivator p300 as one mechanism for competitive
transcriptional regulation of a target gene (24 ). We, therefore,
examined whether p300/CBP, which interacts with both Stats and NF
B,
might be a target of Stat5b inhibition. First, COS cells were
cotransfected with the PRL-R, p300, and the IRF-1-CAT (Fig. 6A
). Exogenous p300 further enhanced PRL
signaling, suggesting that p300 is limiting in the COS transfection
system, and that p300 enhances PRL-inducible Stat1-mediating signaling
to the IRF-1 promoter. To determine whether increased expression of
p300 would reverse Stat5b inhibition, increasing concentrations of p300
were cotransfected with Stat5b and either the 1.7-kb IRF-1 (Fig. 6B
) or
NF
B-TK (Fig. 6C
) promoter. p300 did not affect TNF
signaling to
either the IRF-1 or NF
B-TK promoter. However, p300 reversed
Stat5b-mediated inhibition at the IRF-1 promoter in a dose-dependent
manner, not only in response to PRL but also to PRL plus TNF
stimulation (Fig. 6B
). Similarly, p300 also reversed Stat5b-mediated
inhibition at the NF
B-TK promoter in a dose-dependent manner (Fig. 6C
). These results support our interpretation that Stat5b-mediated
inhibition most likely occurs via a mechanism in which Stat5b
competitively squelches limiting amounts of the coactivator
p300/CBP, and thereby functionally antagonizes Stat1- and
NF
B-mediated signaling to target promoters.
 |
DISCUSSION
|
---|
Stats are a family of latent transcription factors that
participate in cytokine signaling by controlling early gene
transcription. When activated, Stat factors can function to not only
stimulate but also inhibit gene transcription (11 ). Recent studies
suggest that the activity of Stat factors is modulated by their
interaction with other transcription factors (25 ) and/or coactivators
(26 ). These protein-protein interactions lead to synergistic (25 ) or
antagonistic (11 24 25 ) effects on gene transcription. Our previous
observations show that Stat5b inhibits PRL signaling to the IRF-1
promoter (11 ). Our present studies further show that PRL-inducible
Stat5b inhibits NF
B signaling to the IRF-1 promoter and to a TK
promoter containing only NF
B elements. Our studies illustrate that
one mechanism by which Stat5b inhibits transcriptional responses is by
sequestering limiting coactivators at target promoters.
Activation of NF
B, either by overexpression or by TNF
stimulation, can induce IRF-1 promoter activity (Figs. 1
, 2
, 4E
, and 5A
). PRL stimulation further enhances NF
B-mediated induction of the
IRF-1 promoter, presumably due to PRL activation of endogenous Stat1 in
COS cells (Figs. 1
, 2
, 4E
, and 5A
). These results agree with recent
observations that Stat1 and NF
B synergistically activate the IRF-1
promoter, via the GAS and NF
B elements, respectively, in response to
IFN
and TNF
stimulation (1 2 ). Interestingly, both Stat1- and
NF
B-mediated induction of the IRF-1 promoter are inhibited by Stat5b
(Figs. 1
, 2
, 4E
, and 5A
). This Stat5b-mediated inhibition is dependent
upon PRL stimulation, as this leads to Stat5b tyrosine phosphorylation,
dimerization, and nuclear translocation. This inhibitory activity of
Stat5b does not require Stat5b to interact with DNA (Figs. 2
and 3
) but
does require a functional carboxyl terminus (Fig. 5
), presumably so
that Stat5b can engage in protein-protein interaction with a factor
that is required for both Stat1- and NF
B-mediated promoter
activation. This is corroborated by a Stat5b DNA-binding mutant, Stat5b
VVVI, which is as effective as wild-type Stat5b in inhibiting PRL
signaling to the IRF-1 promoter (Figs. 4
and 5
), by Stat5b inhibition
of a mutant IRF-1 promoter that lacks a functional GAS element (Fig. 2
), and by Stat5b inhibition of the TK promoter that contains only
NF
B elements (Fig. 3
). Together, these results strongly support our
interpretation that Stat5b inhibition is mediated by protein-protein
interactions.
What proteins might be the target of Stat5b inhibition? Stat1 does not
directly interact with Stat5 as determined by biacore competition
assays, thus ruling out direct complex formation between Stat1 and
Stat5 (27 ). Although other Stat proteins have been found to
directly interact with NF
B (3 ), Stat5b does not appear to
interact directly with NF
B as assessed by gel shift assays,
glutathione-S-transferase interaction assays, and
coimmunoprecipitation experiments (data not shown) (28 ). Thus, neither
Stat1 nor NFkB are direct targets of Stat5b inhibition at the IRF-1
promoter. Our studies show that the coactivator p300 can functionally
reverse Stat5b inhibition at the IRF-1 and NF
B-TK promoters (Fig. 6
), suggesting that the coactivator p300 is one target of Stat5b
inhibition at these promoters. Multiple contacts sites between Stat1
and p300/CBP (17 ) and between p65 NF
B and CBP (22 23 ) have been
described. Stat5a and Stat1 interact with an overlapping site in p300,
which also interacts with p65 NF
B (21 ), suggesting that competition
for p300 binding might form a basis of their functional antagonism at
the IRF-1 promoter. How the Stats and NF
B interact with p300, which
domains of these proteins are involved, and how their transcriptional
activities are either enhanced or diminished by interactions with p300
are currently under analysis.
The carboxyl terminus of Stat5a is critical for interaction with
p300 to up-regulate the PRL-responsive ß-casein promoter (21 ). Our
studies show that the carboxyl terminus of the highly related Stat5b is
critical in mediating inhibition of the IRF-1 promoter, presumably by
competing with Stat1 for binding to p300. How can these Stat5/p300
interactions be stimulatory at the ß-casein promoter but inhibitory
at the IRF-1 promoter? One major difference is that Stat5b needs to
bind to the ß-casein GAS element for transcriptional induction (11 21 ) while Stat5b does not need to interact with the IRF-1 GAS element
for transcriptional repression (Figs. 2
, 4
, and 5
and Ref. 11 ). How the
coactivator p300/CBP integrates the activities of Stats and other
promoter-specific DNA-binding proteins may be distinct and may
contribute to differences in the transcriptional activities of the
Stat5/p300 complex at the two promoters (26 29 ). Furthermore, recent
studies show that distinct coactivator complexes are recruited by Stat
(17 20 26 ) and by NF
B (30 31 ) to regulate gene transcription. It
is possible that in addition to p300/CBP, other coactivators such as
SRC-3 (29 ) and SRC-1 (30 31 ) may also be involved in PRL-inducible
Stat1 or TNF
-inducible NF
B signaling, respectively, to the IRF-1
promoter. In this regard, it would be interesting to determine whether
SRC-1 in combination with p300 will fully reverse Stat5b inhibition of
NF
B signaling to the NF
B-TK promoter (Fig. 6C
). These
observations support our findings that Stat5b inhibition of Stat1 or
NF
B signaling is not mediated by Stat-DNA interactions but is
mediated by Stat/p300/CBP interactions at the target promoters.
Immunofluorescence studies show that Stat5b is translocated into the
nucleus upon PRL stimulation (Fig. 4B
). In contrast, Stat5b VVVI mutant
remains primarily cytoplasmic and does not accumulate significantly in
the nucleus even after PRL stimulation (Fig. 4D
). The lack of nuclear
accumulation of the Stat5b VVVI mutant explains in part the inability
of Stat5b VVVI mutant to inhibit NF
B signaling to either the IRF-1
promoter (Fig. 4E
) or the NF
B TK promoter (Fig. 4F
). Yet, the Stat5b
VVVI mutant is still capable of inhibiting Stat1-mediated IRF-1
promoter activity (Figs. 4E
and 5A
). Our hypothesis is that Stat5b
inhibition of NF
B signaling requires its nuclear localization.
However, Stat5b inhibition of Stat1 signaling may occur not only at the
transcriptional level, as is the case for wild-type Stat5b, but also at
an extranuclear level for the Stat5b VVVI mutant. A potential mechanism
may be that the Stat5b VVVI mutant competes for a cytoplasmic factor
that can modulate gene transcription in the nucleus. Two such factors
have recently been described. N-myc interacting protein, Nmi (33 ), has
been shown to interact with Stat1 and Stat5, and Nmi-Stat interaction
was shown to stabilize the Stat-CBP complex and to enhance
Stat-mediated gene transcription. The growth factor and cytokine
receptor adaptor protein, CrkL, has been shown to interact with Stat5
and participate in Stat5 binding to DNA (34 35 ). Whether these factors
participate in PRL signaling to the IRF-1 promoter, and whether they
are targets of Stat5b and/or Stat5b VVVI inhibition are currently under
investigation.
Alternatively, Stat5b VVVI mutant may inhibit Stat1 nuclear
translocation and thereby impede Stat1 function at the transcriptional
level. This interpretation is consistent with the observation that
Stat5b VVVI mutant accumulates in the cytoplasm and the perinuclear
region in PRL-stimulated cells and may suggest problems in its
transport across the nuclear membrane (36 37 ). Studies to examine
Stat1 nuclear translocation in the presence of wild-type and Stat5b
VVVI mutant will test this hypothesis. Previous studies have also
demonstrated that the Stat5b VVVI mutant remained cytoplasmic even
after 1 h of GH stimulation of several different cell types (38 )
and is not specific to transfected COS cells.
Together, these studies reveal novel features of Stat regulation
of gene expression. First, Stats can act to repress gene transcription.
Recent studies of the Stat5a/Stat5b double knockout animals suggest
that Stat5 may act as a transcriptional repressor in vivo
(32 ). Second, signaling pathways that activate Stat factors can inhibit
signaling pathways that activate NF
B, depending on the stimuli and
Stat factors involved. For example, Stat1 synergizes while Stat5b
inhibits NF
B signaling to the IRF-1 promoter (this work), and Stat2
inhibits NF
B signaling to the HIV LTR (24 ). Third, Stat regulation
of gene expression may occur not only at the level of gene
transcription (39 ) but also at an extranuclear level, perhaps involving
nuclear transport. This hypothesis is consistent with the observation
that Stat factors reside primarily in the cytoplasm and are activated
to enter the nucleus for a limited time after which they appear to
recycle back into the cytoplasm by the importin
/ß transport
pathway (37 ). Studies are underway to examine the kinetics of nuclear
translocation of Stat5b, Stat1, and NF
B in response to PRL and
TNF
stimulation. The negative cross-talk between Stat5b and NF
B
may elucidate how lactogenic hormones can inhibit TNF
signaling and
provide protection against septic shock (40 ) and hemorrhagic shock
(41 ). These results may also elucidate more general mechanisms
involving competing cytokine regulation of target genes via the
activation of competing Stat factors as occurs during a Th1
vs. Th2 immune response (42 ).
 |
MATERIALS AND METHODS
|
---|
DNA Constructs
The Nb2 PRL-R (pECE), Stat5b, Stat5b
40C, and Stat5bVVVI
mutants [pcDNA3.1(-)], wild-type 1.7-kb IRF-1-CAT, and GAS mutant
1.7-kb IRF-1-CAT constructs were described previously (10 11 43 ).
Briefly, Stat5b
40C is missing the most carboxyl 40 amino acids,
which contain the transactivation domain (gift of Dr. Georg H. Fey,
Friedrich-Alexander University, Erlangen, Germany) (44 ). Stat5b VVVI
was generated by site-directed mutagenesis, in which alanines replaced
the highly conserved VVVI residues in the DNA-binding domain (11 ).
pCMVp65 and pCMVp50 NF
B plasmids were provided by Dr. Tse-Hua Tan
(45 ). p6TKCAT plasmid containing two copies of the NF
B elements was
provided by Dr. Paula M. Pitha (14 ).
Transient Transfection and CAT Assays
COS-1 cells (2 x 105/well) were
seeded in six-well tissue culture plates overnight in DMEM containing
10% FBS (JRH Biosciences, Lenexa, KS) (11 ). Transient
transfections were performed using LipofectAMINE (Life Technologies, Inc., Inc., Gaithersburg, MD) as described
previously (11 ). Plasmid DNA concentrations used for transfection are:
1 µg of Nb2 PRL-R, between 0.05 µg and 0.5 µg of Stat5b,
Stat5b
40C, or Stat5b VVVI, 0.1 µg each of p50 NF
B and p65
NF
B, and 0.1 µg of the various CAT reporter constructs. Empty
vectors (pcDNA3.1 for Stat5 or pCMV for NF
B) were used as controls
as well as for adjusting total DNA concentration in dose-response
experiments. After transfection, cells were maintained for 24 h in
3 ml of DMEM with 1% horse serum (ICN-Flow Laboratories, MacLean, VA)
and were stimulated with either 100 ng/ml ovine PRL (NIDDK-oPRL-20) or
15 ng/ml human TNF
(1 x 107 U/mg) (R&D
Systems Inc., Minneapolis, MN) for 24 h. Cells were lysed in 600
µl/well of reporter lysis buffer (Promega Corp.,
Madison, WI), and 40 µl of cell extracts, 5 µl of 5 mg/ml
n-butyryl-coenzyme A (Promega Corp.), and 3
µl of [14C]chloramphenicol (50 mCi/mmol,
NEN Life Science Products, Boston, MA) were assayed for
4 h at 37 C as described previously (11 ). CAT activity was
analyzed by liquid scintillation counting and normalized to counts per
µg of protein assayed. Each experiment was set up in triplicate.
Error bars represent SEM derived from three
to five independent experiments. Data were plotted by using Origin 4.0
(Microcal Software, Inc., Northampton, MA).
Immunofluorescence
COS cells were cultured on glass coverslips coated with
poly-D-lysine (1 mg/ml, 70,000150,000 Da,
Sigma, St. Louis, MO). Transient transfections were
performed by the calcium phosphate precipitation method using a
mammalian cell transfection kit (Specialty Media Inc., Lavallette, NJ).
PRL-R (2 µg) was cotransfected with either 2 µg of Stat5b or
Stat5b VVVI. After transfection, cells were maintained in DMEM with
1% horse serum for 24 h before stimulation with 100 ng/ml of
PRL for 30 min. The cells were then rinsed twice with ice-cold PBS and
fixed with 4% paraformaldehyde (Polysciences Inc., Warrington, PA) in
PEM buffer (80 mM PIPES, pH 6.9, 1 mM EGTA, 1
mM MgCl2) for 30 min, followed by
permeabilization with 0.5% Triton X-100 in the same buffer for 20 min.
The cells were blocked in 5% milk in TBS-T (20 mM Tris, pH
7.5, 150 mM NaCl, 0.05% Tween-20) containing 0.2% sodium
azide overnight at 4 C, followed by incubation first with
affinity-purified anti-Stat5b antibodies (11 ) at 1:500 dilution for
1 h at room temperature, and then with goat antirabbit IgG
conjugated with Texas Red (Molecular Probes, Inc., Eugene,
OR) at 1:1000 dilution for 30 min. The cells were then washed five
times with TBS-T and stained by 4,6-diamidino-2-phenylindole using
VECTASHIELD mounting media (Vector Laboratories, Inc.,,
Burlingame, CA) on glass slides. Images were obtained using DeltaVision
Deconvolution Confocal Microscopy (Integrated Microscopy Core, Baylor
College of Medicine, Houston, TX).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Tse-Hua Tan for the NF
B constructs, Dr. Paula
Pitha for the 6tk-CAT constructs, Dr. Elena Kabotyanski for assistance
with confocal microscopy, and Dr. Sophia Tsai and Dr. Jeff Rosen for
critical comments.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Li-yuan Yu-Lee, Department of Medicine, Baylor College of Medicine, Houston, Texas 77303.
This work was supported by a Molecular Endocrinology Training Grant
T32-K07696 (G. L.) and by a grant from the NIH RO1-DK-44625 (L.-y.
Y.-L.).
Received for publication March 3, 1999.
Revision received September 15, 1999.
Accepted for publication September 20, 1999.
 |
REFERENCES
|
---|
-
Pine R 1997 Convergence of TNF
and IFN
signaling pathways through synergistic induction of IRF-1/ISGF-2 is
mediated by a composite GAS/
B promoter element. Nucleic Acids Res 25:43464354[Abstract/Free Full Text]
-
Ohmori Y, Schreiber RD, Hamilton TA 1997 Synergy
between interferon-
and tumor necrosis factor-
in
transcriptional activation is mediated by cooperation between signal
transducer and activator of transcription 1 and nuclear factor
B.
J Biol Chem 272:1489914907[Abstract/Free Full Text]
-
Shen C-H, Stavnezer J 1998 Interaction of Stat6 and
NF
B: Direct association and synergistic activation of
interleukin-4-induced transcription. Mol Biol Cell 18:33953430
-
Yu-Lee L-y 1997 Molecular actions of prolactin in the immune
system. Proc Soc Exp Biol Med 215:3552[Abstract]
-
Campbell GS, Argetsinger LS, Ihle JN, Kelly PA, Rillema
JA, Carter-Su C 1994 Activation of JAK2 tyrosine kinase by prolactin
receptors in Nb2 cells and mouse mammary gland explants. Proc Natl Acad
Sci USA 91:52325236[Abstract]
-
Stevens AM, Wang Y, Sieger KA, Lu H, Yu-Lee L-y 1995 Biphasic
transcriptional regulation of the interferon regulatory factor-1 gene
by prolactin: Involvement of gamma-interferon activated sequence and
Stat-related proteins. Mol Endocrinol 9:513525[Abstract]
-
Wang Y, Yu-Lee L-y 1996 Multiple Stat complexes interact at
the IRF-1 GAS in prolactin-stimulated Nb2 T cells. Mol Cell Endocrinol 121:1928[CrossRef][Medline]
-
DaSilva L, Rui H, Erwin RA, Howard OMZ, Kriken RA,
Malabarba MG, Hackett RH, Larner AC, Farrar WL 1996 Prolactin
recruits Stat1, Stat3, and Stat5 independent of conserved receptor
tyrosines Tyr402, Tyr479, Tyr515 and Tyr 580. Mol Cell Endocrinol 117:131140[CrossRef][Medline]
-
Yu-Lee L-y, Hrachovy JA, Stevens AM, Schwarz LA 1990 Interferon-regulatory factor 1 is an immediate-early gene under
transcriptional regulation by prolactin in Nb2 T cells. Mol Cell Biol 10:30873094[Medline]
-
Wang Y, ONeal KD, Yu-Lee L-y 1997 Multiple prolactin
receptor cytoplasmic residues and Stat1 mediate prolactin signaling to
the IRF-1 promoter. Mol Endocrinol 11:13531364[Abstract/Free Full Text]
-
Luo G, Yu-Lee L-y 1997 Transcriptional inhibition by Stat5:
Differential activities at growth-related versus
differentiation-specific promoters. J Biol Chem 272:2684126849[Abstract/Free Full Text]
-
Yu-Lee L-y, Luo G, Moutoussamy S, Finidori J 1998 Prolactin and growth hormone signal transduction in lymphohemopoietic
cells. Cell Mol Life Sci 54:10671075[CrossRef][Medline]
-
Stevens AM, Yu-Lee L-y 1994 Multiple prolactin-responsive
elements mediate G1 and S phase expression of the interferon regulatory
factor-1 gene. Mol Endocrinol 8:345355[Abstract]
-
Vlach J, Pitha PM 1992 Herpes simplex virus type1-mediated
induction of human immunodeficiency virus type 1 provirus correlates
with binding of nuclear proteins to the NF-kappa B enhancer and leader
sequence. J Virol 66:36163623[Abstract]
-
Herrington J, Rui L, Luo G, Yu-Lee L-y, Carter-Su C 1999 A
functional DNA binding domain is required for growth hormone-induced
nuclear accumulation of Stat5b. J Biol Chem 274:51385145[Abstract/Free Full Text]
-
Kazansky AV, Kabotyanski E, Wyszomierski SL, Yel J, Rosen JM 1999 Differential effects of prolactin and src/abl kinase on the
nuclear translocation of Stat5b and Stat5a. J Biol Chem 274:2248422492[Abstract/Free Full Text]
-
Zhang JJ, Vinkemeier U, Gu W, Chakravarti D, Horvath CM,
Darnell Jr JE 1996 Two contact regions between Stat1 and CBP/p300 in
interferon gamma signaling. Proc Natl Acad Sci USA 93:1509215096[Abstract/Free Full Text]
-
Horvai A, Xu L, Korzus E, Brard G, Kalafus D, Mullen T-M, Rose
DW, Rosenfeld MG, Glass CK 1997 Nuclear integration of Jak/Stat and
Ras/AP-1 signaling by CBP and p300. Proc Natl Acad Sci USA 94:10741079[Abstract/Free Full Text]
-
Kurokawa R, Kalafus D, Ogliastro MH, Kioussi C, Xu L, Torchia
J, Rosenfeld MG, Glass CK 1998 Differential use of CREB binding
protein-coactivator complexes. Science 279:700703[Abstract/Free Full Text]
-
Bhattacharya S, Eckner R, Grossman S, Oldread E, Arany Z,
DAndrea A, Livingston DM 1996 Cooperation of Stat2 and p300/CBP in
signalling induced by interferon-
. Nature 383:344347[CrossRef][Medline]
-
Pfitzner E, Jahne R, Wissler M, Stoecklin 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]
-
Gerritsen ME, Williams AJ, Neish AS, Moore S, Shi Y, Collins T 1997 CREB-binding protein/p300 are transcriptional coactivators of p65.
Proc Natl Acad Sci USA 94:29272932[Abstract/Free Full Text]
-
Zhong H, Voll RE, Ghosh S 1998 Phosphorylation of NF
B
p65 by PKA stimulates transcriptional activity by promoting a novel
bivalent interaction with the coactivator CBP/p300. Mol Cell 1:661671[Medline]
-
Hottiger MO, Felzien LK, Nabel GJ 1998 Modulation of
cytokine-induced HIV gene expression by competitive binding of
transcription factors to the coactivator p300. EMBO J 17:31243134[Abstract/Free Full Text]
-
Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional
interactions between Stat5 and the glucocorticoid receptor. Nature 383:726728[CrossRef][Medline]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM,
Mullen T-M, Glass CK, Rosenfeld MG 1998 Transcription factor-specific
requirements for coactivators and their acetyltransferase functions.
Science 279:703707[Abstract/Free Full Text]
-
Greenlund AC, Morales MO, Viviano BL, Yan H, Krolewski J,
Schreiber RD 1995 Stat recruitment by tyrosine-phosphorylated cytokine
receptors: an ordered reversible affinity-driven process. Immunity 2:677687[Medline]
-
Welte T, Leitenberg D, Dittel BN, al-Ramadi BK, Xie B, Chin
YE, Janeway CA, Bothwell ALM, Bottomly K, Fu X-Y 1999 Stat5 interaction
with the T cell receptor complex and stimulation of T cell
proliferation. Science 283:222225[Abstract/Free Full Text]
-
Perissi V, Dasen JS, Kurokawa R, Wang Z, Korzus E, Rose DW,
Glass CK, Rosenfeld MG 1999 Factor-specific modulation of CREB-binding
protein acetyltransferase activity. Proc Natl Acad Sci USA 96:36523657[Abstract/Free Full Text]
-
Sheppard KA, Rose DW, Haque ZK, Kurokawa R, Inerney EM, Westin
S, Thanos D, Rosenfeld MG, Glass CK, Collins T 1999 Transcriptional
activation by NF
B requires multiple coactivators. Mol Cell Biol 19:63676378[Abstract/Free Full Text]
-
Na S-Y, Lee S-K, Han S-J, Choi H-S, Im SY, Lee JW 1998 Steroid
receptor coactivator-1 interacts with the p50 subunit and coactivates
nuclear factor
B-mediated transactivations. J Biol Chem 273:1083110834[Abstract/Free Full Text]
-
Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D,
Wang D, Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b
proteins have essential and nonessential, or redundant, roles in
cytokine responses. Cell 93:841850[Medline]
-
Zhu M, John S, Berg M, Leonard WJ 1999 Functional association
of Nmi with Stat5 and Stat1 in IL-2- and IFN
-mediated
signaling. Cell 96:121130[Medline]
-
Fish EN, Uddin S, Korkmaz M, Majchrzak B, druker BJ, Platanias
LC 1999 Activation of a CrkL-Stat5 signaling complex by type I
interferons. J Biol Chem 274:571573[Abstract/Free Full Text]
-
Ota J, Kimura F, Sato K, Wakimoto N, Nakamura Y, Nagata N,
Suzu S, Yamada M, Shimamura S, Motoyoshi K 1998 Association of CrkL
with Stat5 in hematopoietic cells stimulated by granulocyte-macrophage
colony-stimulating factor or erythropoietin. Biochem Biophys Res Commun 252:779786[CrossRef][Medline]
-
Moore MS 1998 Ran and nuclear transport. J Biol Chem 273:2285722860[Free Full Text]
-
Sekimoto T, Imamoto N, Makajima K, Hirano T, Yoneda Y 1997 Extracellular signal-dependent nuclear import of Stat1 is mediated
by nuclear pore targeting complex formation with NPI-1, but not Rch1.
EMBO J 16:70677077[Abstract/Free Full Text]
-
Struman I, Bentzien F, Lee H, Infroid V, Angelo G, Goffin V,
Weiner RI, Martial JA 1999 Opposing actions of intact and N-terminal
fragments of the human prolactin/growth hormone family members on
angiogenesis: an efficient mechanism of the regulation of angiogenesis.
Proc Natl Acad Sci USA 96:12461251[Abstract/Free Full Text]
-
Darnell JE, Jr 1997 Stats and gene regulation. Science 277:15301635[CrossRef]
-
Gonzalo JA, Mazuchelli R, Mellado M, Frade JMR, Carrera AC,
von Kobbe C, Merida I, Martinez- AC 1996 Enterotoxin septic shock
protection and deficient T helper 2 cytokine production in growth
hormone transgenic mice. J Immunol 157:32983304[Abstract]
-
Zellweger R, Zhu X-H, Wichmann MW, Ayala A, DeMaso CM, Chaudry
IH 1996 Prolactin administration following hemorrhagic shock improves
macrophage cytokine release capacity and decreases mortality from
subsequent sepsis. J Immunol 157:57485754[Abstract]
-
Romagnani S 1997 The Th1/Th2 paradigm. Immunol Today 18:263266[CrossRef][Medline]
-
ONeal KD, Yu-Lee L-y 1994 Differential signal transduction
of the short, Nb2, and long PRL receptor: activation of IRF-1 and cell
proliferation. J Biol Chem 269:2607626082[Abstract/Free Full Text]
-
Ripperger JA, Fritz S, Richter K, Hocke GM, Lottspeich F, Fey
GH 1995 Transcription factors Stat3 and Stat5b are present in rat liver
nuclei late in an acute phase response and bind interleukin-6 response
elements. J Biol Chem 270:2999830006[Abstract/Free Full Text]
-
Lai JH, Horvath G, Subleski J, Bruder J, Ghosh P, Tan T-H 1995 RelA is a potent transcriptional activator of the CD28 response element
within the interleukin 2 promoter. Mol Cell Biol 15:42604271[Abstract]