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B-Mediated Signaling
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NFB) signaling to
both the interferon regulatory factor-1 promoter and to the
thymidine kinase promoter containing multimerized NFB elements
(NFB-TK). Further, PRL-inducible Stat5b inhibits tumor necrosis
factor-
signaling presumably by inhibiting endogenous NFB. This
Stat5b-mediated inhibitory effect on NFB 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
NFB-mediated activation of target promoters. Increasing
concentrations of the coactivator p300/CBP reverses Stat5b inhibition
at both the interferon-regulatory factor-1 and NFB-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 NFB 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 |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B). Although their activation pathways are different, activated
Stats and NFB 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 NFB, are present in the IRF-1 promoter
(13 ). Recent studies have shown that IFN-activated Stat1 and tumor
necrosis factor- (TNF)-activated NFB 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 NFB signaling pathway. In
this report, we examined the negative cross-talk between Stat5b and
NFB signaling at the IRF-1 promoter. We show that Stat5b inhibits
p50/p65 NFB signaling to the IRF-1 promoter as well as to the
heterologous thymidine kinase (TK) promoter containing multimerized
NFB 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 NFB
signaling by competing for coactivators necessary for NFB-mediated
gene transcription.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B Induction of the IRF-1 PromoterB, 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 NFB 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 NFB signaling to the
IRF-1 promoter, COS cells were cotransfected with the PRL-R, Stat5b,
p50 NFB, p65 NFB, 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 NFB 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 NFB, presumably due to the activation of endogenous Stat1
(10 ). In the absence of the PRL stimulation, overexpression of Stat5b
did not affect NFB-mediated induction of the IRF-1 promoter. In
contrast, upon PRL stimulation, Stat5b strongly inhibited both PRL and
NFB-mediated IRF-1 promoter activity in a dose-dependent manner
(Fig. 1). These studies show that NFB can activate the IRF-1
promoter and this induction is inhibited by Stat5b in a dose-dependent
as well as PRL-dependent manner.
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B
Signaling to the IRF-1 PromoterB 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 NFB-mediated activation of the IRF-1 promoter,
confirming that NFB activates the IRF-1 promoter independently of a
functional GAS element. This NFB-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 NFB
signaling to the IRF-1 promoter does not require Stat5b to interact
with the GAS element.
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B Activation of the Heterologous NFB-TK
PromoterB elements,
the heterologous TK promoter containing two copies of the NFB
element was examined (14 ). As expected, the NFB-TK promoter was not
responsive to PRL stimulation, as it does not have a GAS element (Fig. 3). p50/p65 NFB mediated greater than
10-fold induction of the NFB-TK promoter, which again is not further
inducible by PRL stimulation. Interestingly, Stat5b inhibited
the NFB-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 NFB signaling to promoters that
do not contain a GAS element. These results further support our
hypothesis that Stat5b-mediated inhibition of NFB signaling is not
mediated by Stat-DNA interactions but is mediated by protein-protein
interactions.
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B-mediated
signaling to both the IRF-1 and NFB TK promoters, a Stat5b DNA
binding mutant, Stat5b VVVI (11 ), was tested for its ability to inhibit
NFB 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.
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B signaling to either
the IRF-1 (Fig. 4E
) or NFB-TK (Fig. 4F) promoter. Thus, wild-type
Stat5b still inhibits NFB signaling to both the IRF-1 and NFB TK
promoters (Fig. 4, E and F), but the Stat5b VVVI mutant fails to
inhibit NFB 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 NFB (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 NFB 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 NFB
activity as stimulated via TNF, in the absence of p50/p65 NFB
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 NFB 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 NFB 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 NFB signaling to
the IRF-1 promoter. Furthermore, PRL-inducible Stat5b also
inhibited the large 7- to 8-fold TNF-inducible NFB signaling to
the heterologous NFB-TK promoter (Fig. 5B).
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40C mutant, which lacks
the carboxyl-terminal 40 amino acids, fails to inhibit Stat1-mediated
PRL signaling to the IRF-1 promoter (11 ). The Stat5b40C mutant also
failed to inhibit TNF signaling to both the IRF-1 (Fig. 5A) and
NFB-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 NFB-TK (Fig. 5B) promoter. In agreement with
the data on overexpression of p50/p65 NFB (Fig. 4), these results
suggest that Stat5b inhibits TNF signaling by inhibiting endogenous
NFB, 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 NFB (22 23 24 ) to enhance target gene expression. For example,
IFN-inducible Stat2 appears to compete with TNF-inducible NFB
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 NFB,
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
NFB-TK (Fig. 6C) promoter. p300 did not affect TNF signaling to
either the IRF-1 or NFB-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 NFB-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
NFB-mediated signaling to target promoters.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B signaling to the IRF-1 promoter and to a TK
promoter containing only NFB elements. Our studies illustrate that
one mechanism by which Stat5b inhibits transcriptional responses is by
sequestering limiting coactivators at target promoters.
Activation of NFB, either by overexpression or by TNF
stimulation, can induce IRF-1 promoter activity (Figs. 1, 2, 4E, and 5A). PRL stimulation further enhances NFB-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 NFB synergistically activate the IRF-1
promoter, via the GAS and NFB elements, respectively, in response to
IFN and TNF stimulation (1 2 ). Interestingly, both Stat1- and
NFB-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 NFB-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
NFB 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 NFB (3 ), Stat5b does not appear to
interact directly with NFB 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 NFB-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 NFB and CBP (22 23 ) have been
described. Stat5a and Stat1 interact with an overlapping site in p300,
which also interacts with p65 NFB (21 ), suggesting that competition
for p300 binding might form a basis of their functional antagonism at
the IRF-1 promoter. How the Stats and NFB 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 NFB (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 NFB 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
NFB signaling to the NFB-TK promoter (Fig. 6C). These
observations support our findings that Stat5b inhibition of Stat1 or
NFB 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 NFB signaling to either the IRF-1
promoter (Fig. 4E) or the NFB 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 NFB 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 NFB, depending on the stimuli and
Stat factors involved. For example, Stat1 synergizes while Stat5b
inhibits NFB signaling to the IRF-1 promoter (this work), and Stat2
inhibits NFB 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 NFB in response to PRL and
TNF stimulation. The negative cross-talk between Stat5b and NFB
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 ).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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, Stat5b40C 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 NFB plasmids were provided by Dr. Tse-Hua Tan
(45 ). p6TKCAT plasmid containing two copies of the NFB 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,
Stat5b40C, or Stat5b VVVI, 0.1 µg each of p50 NFB and p65
NFB, and 0.1 µg of the various CAT reporter constructs. Empty
vectors (pcDNA3.1 for Stat5 or pCMV for NFB) 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, 70,000–150,000 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).
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ACKNOWLEDGMENTS |
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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 |
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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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and IFN
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p65 by PKA stimulates transcriptional activity by promoting a novel
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B requires multiple coactivators. Mol Cell Biol 19:6367–6378B-mediated transactivations. J Biol Chem 273:10831–10834-mediated
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 X Han, X Ren, I Jurickova, K Groschwitz, B A Pasternak, H Xu, T A Wilson, S P Hogan, and L A Denson Regulation of intestinal barrier function by signal transducer and activator of transcription 5b Gut, January 1, 2009; 58(1): 49 - 58. [Abstract] [Full Text] [PDF]
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 M. D. Buzzelli, M. Nagarajan, J. F. Radtka, M. L. Shumate, M. Navaratnarajah, C. H. Lang, and R. N. Cooney Nuclear Factor-{kappa}B Mediates the Inhibitory Effects of Tumor Necrosis Factor-{alpha} on Growth Hormone-Inducible Gene Expression in Liver Endocrinology, December 1, 2008; 149(12): 6378 - 6388. [Abstract] [Full Text] [PDF]
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 R. N. Dhir, C. Thangavel, and B. H. Shapiro Attenuated Expression of Episodic Growth Hormone-Induced CYP2C11 in Female Rats Associated with Suboptimal Activation of the Jak2/Stat5B and Other Modulating Signaling Pathways Drug Metab. Dispos., November 1, 2007; 35(11): 2102 - 2110. [Abstract] [Full Text] [PDF]
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 M. Ono, D. J. Chia, R. Merino-Martinez, A. Flores-Morales, T. G. Unterman, and P. Rotwein Signal Transducer and Activator of Transcription (Stat) 5b-Mediated Inhibition of Insulin-Like Growth Factor Binding Protein-1 Gene Transcription: A Mechanism for Repression of Gene Expression by Growth Hormone Mol. Endocrinol., June 1, 2007; 21(6): 1443 - 1457. [Abstract] [Full Text] [PDF]
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 J. C. Choy, Y. Wang, G. Tellides, and J. S. Pober Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature PNAS, January 23, 2007; 104(4): 1313 - 1318. [Abstract] [Full Text] [PDF]
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D. J. Waxman and C. O'Connor Growth Hormone Regulation of Sex-Dependent Liver Gene Expression Mol. Endocrinol., November 1, 2006; 20(11): 2613 - 2629. [Abstract] [Full Text] [PDF]
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 K. E. Jie, M. C. Verhaar, M.-J. M. Cramer, K. van der Putten, C. A. J. M. Gaillard, P. A. Doevendans, H. A. Koomans, J. A. Joles, and B. Braam Erythropoietin and the cardiorenal syndrome: cellular mechanisms on the cardiorenal connectors Am J Physiol Renal Physiol, November 1, 2006; 291(5): F932 - F944. [Abstract] [Full Text] [PDF]
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 T. Ahmed, G. Yumet, M. Shumate, C. H. Lang, P. Rotwein, and R. N. Cooney Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G35 - G44. [Abstract] [Full Text] [PDF]
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K. H. Clodfelter, M. G. Holloway, P. Hodor, S.-H. Park, W. J. Ray, and D. J. Waxman Sex-Dependent Liver Gene Expression Is Extensive and Largely Dependent upon Signal Transducer and Activator of Transcription 5b (STAT5b): STAT5b-Dependent Activation of Male Genes and Repression of Female Genes Revealed by Microarray Analysis Mol. Endocrinol., June 1, 2006; 20(6): 1333 - 1351. [Abstract] [Full Text] [PDF]
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 J. Jensen, E. D Galsgaard, A. E Karlsen, Y. C Lee, and J. H Nielsen STAT5 activation by human GH protects insulin-producing cells against interleukin-1{beta}, interferon-{gamma} and tumour necrosis factor-{alpha}-induced apoptosis independent of nitric oxide production J. Endocrinol., October 1, 2005; 187(1): 25 - 36. [Abstract] [Full Text] [PDF]
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T. Matsuda, K. Ferreri, I. Todorov, Y. Kuroda, C. V. Smith, F. Kandeel, and Y. Mullen Silymarin Protects Pancreatic {beta}-Cells against Cytokine-Mediated Toxicity: Implication of c-Jun NH2-Terminal Kinase and Janus Kinase/Signal Transducer and Activator of Transcription Pathways Endocrinology, January 1, 2005; 146(1): 175 - 185. [Abstract] [Full Text] [PDF]
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 A. M. Corbacho, G. Valacchi, L. Kubala, E. Olano-Martin, B. C. Schock, T. P. Kenny, and C. E. Cross Tissue-specific gene expression of prolactin receptor in the acute-phase response induced by lipopolysaccharides Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E750 - E757. [Abstract] [Full Text] [PDF]
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 J. F. Challacombe, A. Rechtsteiner, R. Gottardo, L. M. Rocha, E. P. Browne, T. Shenk, M. R. Altherr, and T. S. Brettin Evaluation of the host transcriptional response to human cytomegalovirus infection Physiol Genomics, June 17, 2004; 18(1): 51 - 62. [Abstract] [Full Text] [PDF]
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 Z. Yu and B. C. Kone The STAT3 DNA-Binding Domain Mediates Interaction with NF-{kappa}B p65 and Inducible Nitric Oxide Synthase Transrepression in Mesangial Cells J. Am. Soc. Nephrol., March 1, 2004; 15(3): 585 - 591. [Abstract] [Full Text] [PDF]
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 M. R. Horton, S. Boodoo, and J. D. Powell NF-kappa B Activation Mediates the Cross-talk between Extracellular Matrix and Interferon-gamma (IFN-gamma ) Leading to Enhanced Monokine Induced by IFN-gamma (MIG) Expression in Macrophages J. Biol. Chem., November 8, 2002; 277(46): 43757 - 43762. [Abstract] [Full Text] [PDF]
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 M. M. Faraldo, M.-A. Deugnier, S. Tlouzeau, J. P. Thiery, and M. A. Glukhova Perturbation of beta 1-Integrin Function in Involuting Mammary Gland Results in Premature Dedifferentiation of Secretory Epithelial Cells Mol. Biol. Cell, October 1, 2002; 13(10): 3521 - 3531. [Abstract] [Full Text] [PDF]
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Y. E. Timsit and D. S. Riddick Stimulation of Hepatic Signal Transducer and Activator of Transcription 5b by GH Is Not Altered by 3-Methylcholanthrene Endocrinology, September 1, 2002; 143(9): 3284 - 3294. [Abstract] [Full Text] [PDF]
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 I. Y. H. Mak, J. J. Brosens, M. Christian, F. A. Hills, L. Chamley, L. Regan, and J. O. White Regulated Expression of Signal Transducer and Activator of Transcription, Stat5, and its Enhancement of PRL Expression in Human Endometrial Stromal Cells in Vitro J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2581 - 2588. [Abstract] [Full Text] [PDF]
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R.-C. Wu, J. Qin, Y. Hashimoto, J. Wong, J. Xu, S. Y. Tsai, M.-J. Tsai, and B. W. O'Malley Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator Activity by I{kappa}B Kinase Mol. Cell. Biol., May 15, 2002; 22(10): 3549 - 3561. [Abstract] [Full Text] [PDF]
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T. Nakamura, R. Ouchida, T. Kodama, T. Kawashima, Y. Makino, N. Yoshikawa, S. Watanabe, C. Morimoto, T. Kitamura, and H. Tanaka Cytokine Receptor Common beta Subunit-mediated STAT5 Activation Confers NF-kappa B Activation in Murine proB Cell Line Ba/F3 Cells J. Biol. Chem., February 15, 2002; 277(8): 6254 - 6265. [Abstract] [Full Text] [PDF]
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 T. L.-Y. Chang, A. Mosoian, R. Pine, M. E. Klotman, and J. P. Moore A Soluble Factor(s) Secreted from CD8+ T Lymphocytes Inhibits Human Immunodeficiency Virus Type 1 Replication through STAT1 Activation J. Virol., January 15, 2002; 76(2): 569 - 581. [Abstract] [Full Text] [PDF]
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 L.-Y. Yu-Lee Prolactin Modulation of Immune and Inflammatory Responses Recent Prog. Horm. Res., January 1, 2002; 57(1): 435 - 455. [Abstract] [Full Text] [PDF]
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S.-H. Park and D. J. Waxman Inhibitory Cross-talk between STAT5b and Liver Nuclear Factor HNF3beta . IMPACT ON THE REGULATION OF GROWTH HORMONE PULSE-STIMULATED, MALE-SPECIFIC LIVER CYTOCHROME P-450 GENE EXPRESSION J. Biol. Chem., November 9, 2001; 276(46): 43031 - 43039. [Abstract] [Full Text] [PDF]
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 L.-y. Yu-Lee Stimulation of interferon regulatory factor-1 by prolactin Lupus, October 1, 2001; 10(10): 691 - 699. [Abstract] [PDF]
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A. Flores-Morales, L. Fernandez, E. Rico-Bautista, A. Umana, C. Negrin, J.-G. Zhang, and G. Norstedt Endoplasmic Reticulum Stress Prolongs GH-Induced Janus Kinase (JAK2)/Signal Transducer and Activator of Transcription (STAT5) Signaling Pathway Mol. Endocrinol., September 1, 2001; 15(9): 1471 - 1483. [Abstract] [Full Text] [PDF]
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 J. G. Bode, R. Fischer, D. Haussinger, L. Graeve, P. C. Heinrich, and F. Schaper The Inhibitory Effect of IL-1{beta} on IL-6-Induced {alpha}2-Macroglobulin Expression Is Due to Activation of NF-{kappa}B J. Immunol., August 1, 2001; 167(3): 1469 - 1481. [Abstract] [Full Text] [PDF]
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W. K. Shea-Eaton, P.-P. H. Lee, and M. M. Ip Regulation of Milk Protein Gene Expression in Normal Mammary Epithelial Cells by Tumor Necrosis Factor Endocrinology, June 1, 2001; 142(6): 2558 - 2568. [Abstract] [Full Text] [PDF]
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D. M. Stewart, G. A. Johnson, C. A. Vyhlidal, R. C. Burghardt, S. H. Safe, L.-Y. Yu-Lee, F. W. Bazer, and T. E. Spencer Interferon-{{tau}} Activates Multiple Signal Transducer and Activator of Transcription Proteins and Has Complex Effects on Interferon-Responsive Gene Transcription in Ovine Endometrial Epithelial Cells Endocrinology, January 1, 2001; 142(1): 98 - 107. [Abstract] [Full Text] [PDF]
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B. N. Friedrichsen, E. D. Galsgaard, J. H. Nielsen, and A. Møldrup Growth Hormone- and Prolactin-Induced Proliferation of Insulinoma Cells, INS-1, Depends on Activation of STAT5 (Signal Transducer and Activator of Transcription 5) Mol. Endocrinol., January 1, 2001; 15(1): 136 - 148. [Abstract] [Full Text]
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H. K. Choi and D. J. Waxman Plasma Growth Hormone Pulse Activation of Hepatic JAK-STAT5 Signaling: Developmental Regulation and Role in Male-Specific Liver Gene Expression Endocrinology, September 1, 2000; 141(9): 3245 - 3255. [Abstract] [Full Text] [PDF]
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Y. Ohmori and T. A. Hamilton Interleukin-4/STAT6 Represses STAT1 and NF-kappa B-dependent Transcription through Distinct Mechanisms J. Biol. Chem., November 22, 2000; 275(48): 38095 - 38103. [Abstract] [Full Text] [PDF]
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