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Department of Cell Biology (S.L.W., J.M.R.) Baylor College of
Medicine Houston Texas 77030-3498
University of Texas
Health Science Center (J.Y.) San Antonio, Texas 78284
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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After HC uptake into cells, it binds to intracellular GR. Ligand binding induces a conformational change in GR and dissociation of heat shock proteins, which are associated with the latent form of the receptor (reviewed in Ref. 3). Unlike many steroid receptors, GR resides primarily in the cytoplasm in the absence of ligand. Upon ligand binding, it translocates to the nucleus (4), where it binds to palindromic glucocorticoid response elements (GREs) in the promoters of target genes, as well as interacts with GRE half-sites and other trans-acting factors (reviewed in Refs. 5, 6).
PRL binds to the extracellular portion of the PRL receptor (PRL-R) and
initiates events in the JAK/STAT signal transduction cascade (7).
Specifically, JAK2, which is associated with the PRL-R in the absence
of ligand, is activated by transphosphorylation when two JAK2
molecules are brought together by ligand-induced dimerization of
the receptor (8). The activated JAK2 tyrosine phosphorylates PRL-R,
creating docking sites for SH2 domain-containing proteins, including
STAT5 (9), which subsequently are tyrosine phosphorylated, dimerize,
and then translocate to the nucleus. Activated STAT5 binds to DNA sites
known as GAS elements (named for 
-interferon-activated sequences)
and modulates the activity of target genes containing GAS elements in
their promoters, such as the ß-casein gene (10, 11).
STAT5 was first identified as a binding activity in tissue extracts
from lactating mammary gland and was referred to as MGF (mammary gland
factor) (12, 13, 14). Molecular cloning revealed that MGF was in fact STAT5
(11) and was neither lactation specific nor mammary gland specific (11, 15, 16). In fact, STAT5 was activated by many hormones, growth factors,
and cytokines, including PRL (11, 17, 18, 19, 20, 21, 22, 23). Two clustered STAT5 genes,
STAT5a and STAT5b, have been identified that are more than 90%
identical and probably arose by gene duplication (16). Both STAT5a and
STAT5b encode different isoforms, some of which may arise by
alternative splicing. In particular, carboxy-truncated isoforms are
common for the STAT family of transcription factors (15, 24, 25, 26, 27, 28). A
carboxy-truncated STAT5a isoform, designated STAT5a2, was first
identified while screening a cDNA library prepared from RNA isolated
from the rat mammary gland at day 2 of lactation. The mRNA for STAT5a2
is generated by a 1.7-kb insertion that encodes a stop codon resulting
in a 53-amino acid deletion (15). A very similar alternative splice
form, called STAT5b
40C, was identified for STAT5b. STAT5b40C is
truncated at the same position in the protein; however, only 40 amino
acids are deleted due to differences in the carboxy-terminal
sequences of STAT5a and STAT5b (25). These naturally occurring
carboxy-truncated STAT5 isoforms may act as dominant negative
inhibitors of STAT5-dependent transcription and cannot independently
activate transcription because they lack the carboxy-terminal
transactivation domain. They remain tyrosine phosphorylated and bound
to GAS sites for longer periods of time than full-length STAT5 isoforms
after PRL treatment, suggesting that the carboxy-terminal
sequences may affect the interaction with a tyrosine phosphatase (28, 29).
STAT5 and GR have been shown to interact, both in transiently transfected COS cells (2) and in mammary epithelial cells (30). In the HC11 mammary epithelial cell line, STAT5 and GR are associated independently of HC and PRL treatment. This association was also observed in tissue extracts prepared at all stages of mammary gland development (30). STAT5 and GR activate transcription from the ß-casein promoter in a synergistic fashion in transiently transfected COS cells (2), and both STAT5a and STAT5b synergize with GR (31). The C-terminal transactivation domain of STAT5 is not necessary for this transcriptional synergy (31, 32), but the amino-terminal TAF-1 domain of GR is required (31). One mechanism proposed for STAT5/GR transcriptional synergy is that STAT5 recruits GR to the promoter and allows the strong transactivation domain of GR to supplement the weaker transactivation domain of STAT5.
We initiated experiments to better understand the mechanism of transcriptional synergy between STAT5 and GR. Using electrophoretic mobility shift assays (EMSAs), immunoprecipitation, and Western blotting at various times after PRL treatment, it was discovered that GR both enhanced and prolonged the DNA-binding activity of STAT5. This was correlated with increased STAT5 tyrosine phosphorylation suggesting, therefore, that GR enhances STAT5 DNA binding by modulating the phosphorylation state of STAT5. That this enhancement may be specific to GR was demonstrated in experiments where the estrogen receptor (ER) exerted the opposite effect. Enhancement of STAT5 activity by GR is, therefore, one component of the transcriptional synergy exhibited by STAT5 and GR at the ß-casein promoter. A similar mechanism may be operable at other STAT5-dependent promoters.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and GR
(Fig. 1B) are both predominately localized in the cytoplasm. PRL
treatment results in the nuclear localization of STAT5a (Fig. 1C), and
HC treatment translocates GR to the nucleus (Fig. 1F) as expected. In
cotransfected cells treated with only PRL, a subset of the GR in the
cell is present in the nucleus in the absence of any HC stimulation
(Fig. 1D). GR does not translocate to the nucleus after PRL treatment
in the absence of STAT5 cotransfection (data not shown). The converse
was also observed. In cells treated with only HC, a portion of the
STAT5 is localized in the nucleus (Fig. 1E), and no translocation of
STAT5 is seen in response to HC in the absence of GR (data not shown).
Thus, PRL-activated STAT5 can interact with and translocate GR into the
nucleus, and conversely ligand-bound GR can interact with and
translocate inactivated STAT5 into the nucleus. The ability of STAT5
and GR to translocate in the nucleus after only one stimulus indicates
an interaction between the two proteins. Colocalization of some of the
nuclear STAT5 and GR was demonstrated by double IIF using deconvolution
confocal microscopy (data not shown).
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, lanes 2 and 6). This complex can be
almost completely supershifted with antibody to STAT5a (Fig. 2C, lane
4). The GR-dependent increase in STAT5a DNA binding was not due to an
increase in STAT5a protein (Fig. 2B). Cotransfection of GR without HC
treatment resulted in an intermediate level of STAT5 DNA binding (Fig. 2A, lane 4). GR and HC had no detectable effect
on the DNA binding of STAT5a in the absence of PRL (Fig. 2A, lanes 3
and 5). GR enhanced the DNA-binding activity of STAT5b in a similar
fashion to that of STAT5a (data not shown).
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, lane 4) and
in light of data from other laboratories concerning STAT5-GR
protein-protein interactions (2, 30), it is likely that GR enhancement
of STAT5 DNA binding represents a direct effect of GR. Therefore,
STAT5a DNA binding was examined after concurrent treatment with PRL and
HC. A similar increase in STAT5a DNA binding was seen when HC and PRL
were added concurrently for 30 min as compared with pretreatment
overnight with HC followed by 30 min of PRL treatment (Fig. 2C, compare
lanes 2 and 3). In COS-1 cells, the time of HC treatment did not
influence the increase in STAT5a DNA binding imparted by GR. This is
probably due to overexpression of STAT5 and GR in these cells. In other
cell lines, pretreatment with HC was not sufficient to see an increase
in STAT5a DNA binding (see below). These results suggest that
enhancement of STAT5 DNA binding is a direct rather than an indirect
effect of GR. The GR-dependent increase in STAT5a DNA binding was also
observed when an oligonucleotide with both 1/2 GREs mutated was used as
the probe (Fig. 2D). Thus, despite the presence of 1/2 GREs in the
oligonucleotide used for EMSA, the principal GR-enhanced STAT5a complex
detected in these experiments most likely does not contain stably
associated GR (see Discussion).
Because COS-1 cells are transformed with SV40 large T antigen and
amplify plasmids containing an SV40 origin of replication, we wanted to
ensure that GR enhancement of STAT5 DNA binding was not due only to
overexpression. Accordingly, CHOk1 cells were transiently transfected
with PRL-R, STAT5a, and GR, and PRL and HC treatment was performed as
indicated. Treatment with PRL + HC for 1.5 h gave a higher
level of STAT5a DNA binding than treatment with PRL alone (Fig. 3, lane B vs. lane C). These
data confirm that GR enhancement of STAT5 DNA binding is observed in
transiently transfected cells in the absence of overexpression. In
CHOk1 cells, GR enhancement of STAT5a DNA binding was seen only with
concurrent PRL and HC treatment and was not observed with overnight HC
treatment before PRL treatment (Fig. 3, lanes C vs. D). This
is in contrast to the situation in COS cells and supports the
theory that enhancement of STAT5 DNA binding by GR is a direct effect
of the STAT5/GR protein-protein interaction.
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, lanes 2–5 and 9–12). This was
particularly evident when the unlabeled oligonucleotide was present at
a 2-fold molar excess or less (Fig. 4A, lanes 2, 3, and 4
vs. lanes 9, 10, and 11). These differences are summarized
quantitatively in Fig. 4B. The ability of the GR- enhanced complex to
more effectively resist competition by an unlabeled oligonucleotide
suggests that the GR-STAT5a interaction results in either a higher DNA
affinity or a slower dissociation rate of STAT5 from the DNA or
both.
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). In the experiment shown in Fig. 5A, the GR-induced increase in STAT5a DNA binding after 30 min of PRL
treatment was less than average: only 20% increase (Fig. 5A, lane 3
compared with lane 4). However, the differences between STAT5 alone and
STAT5 and GR in combination were more pronounced at all the other time
points in the experiment (Fig. 5A, lanes 5–12). Quantitation from
several independent experiments is shown in Fig. 5B. The differences at
1.5 h and 3.0 h after PRL treatment were particularly
striking. After 1.5 h of PRL treatment, the level of STAT5 DNA
binding was approximately 60% of that seen after 30 min of PRL
treatment. When GR was cotransfected and treatment with both HC and PRL
was performed, 94% of the STAT5a DNA-binding activity detected after
30 min of hormonal treatment was still detected at 1.5 h. After
3 h of PRL treatment, the DNA-binding activity in cells
transfected with STAT5a alone decreased to approximately 50% of the
level seen after 30 min of PRL treatment. Cells cotransfected with GR
and treated with both hormones still retained 80% of their original
DNA-binding activity. The differences seen between the two treatment
groups were statistically significant for all four time points tested
(P < 0.05).
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, A and B) and anti-STAT5a C-terminal
antibody (Fig. 6, C and D). When STAT5a alone was transfected, the
level of STAT5a tyrosine phosphorylation was maximal after 30 min of
PRL treatment and decreased rapidly thereafter (Fig. 6A). A faint
complex could still be detected after 1.5 h of PRL treatment in
longer film exposures (Fig. 6A, lane 4; data not shown). No tyrosine
phosphorylation of STAT5 could be detected 3 h after PRL
treatment. The slight discrepancy between detectable tyrosine
phosphorylation and detectable STAT5 DNA binding is explained by the
fact that EMSA is a much more sensitive assay than Western blotting of
immunoprecipitated proteins. When STAT5a and GR were cotransfected and
treatment with both PRL and HC was performed, STAT5a remained tyrosine
phosphorylated for a prolonged time period after PRL treatment (Fig. 6B). The levels of total STAT5a protein did not change significantly
over the course of the experiment (Fig. 6, C and D). The same control
sample (labeled C) was included on all Western blots. Although the
signal intensity in Fig. 6C is less than the signal intensity in Fig. 6D, the intensity of this positive control (Fig. 6, panels C and D,
lanes 8 and 16) is also decreased. The apparent difference is,
therefore, caused by variation in the chemiluminence detection system
and not by a difference in STAT5a protein levels. These results
indicate that the protein-protein interaction between STAT5 and GR
prolongs STAT5 tyrosine phosphorylation and DNA binding after PRL
treatment. This enhancement of STAT5 activity by GR is probably a
contributing factor in the transcriptional synergy exhibited by STAT5
and GR.
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750, an artificially generated construct,
which has been extensively studied (29) (S. L. Wyszomierski and
J. M. Rosen, unpublished results). The transactivation domain of
STAT5a is missing in both of these proteins, and this region has been
postulated to be important for interaction with a tyrosine phosphatase.
When GR is cotransfected, carboxy-truncated STAT5 is converted from a
dominant negative factor into a positive transactivator. Therefore, the
carboxy-terminal region of STAT5 is not required for transcriptional
synergy with GR (31, 32). We were interested in examining whether GR
could also influence the DNA-binding activity of STAT5a2. COS-1 cells
were transiently transfected with PRL-R and STAT5a2, or PRL-R, STAT5a2,
and GR, and cells were treated with HC and PRL as indicated. Unlike
STAT5a, STAT5a2 exhibits a basal level of DNA binding in the absence of
PRL (Fig. 7, lane A). This low level of
STAT5a2 DNA binding in the absence of PRL was dramatically enhanced
almost 10-fold when GR was cotransfected (Fig. 7, lane C) and was
further enhanced by 100% when treatment with HC was performed (Fig. 7, lane E). STAT5a2 exhibited a very high level of DNA binding after 30
min PRL treatment (Fig. 7, lane B). The level of STAT5a2 binding after
PRL treatment was increased when GR was cotransfected to twice the
level seen with STAT5a2 alone (Fig. 7, lane D). Treatment with both HC
and PRL did not lead to an additional enhancement of STAT5a2 binding
(Fig. 7, lane F). This may be because the DNA-binding activity was
already maximal and could not be further increased. Carboxy-truncated
STAT5b behaved in a similar manner (data not shown). Therefore,
carboxy-terminal sequences of STAT5 that are not required for GR-STAT5
transcriptional synergy are also not required for GR enhancement of
STAT5 DNA binding.
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, compare lanes 4 and 6 with lanes 3
and 5). STAT5a DNA binding was decreased by ER even in the absence of
E2 (Fig. 8A, lane 7) similar to the ligand-independent
stimulatory effect of GR. ER did not affect STAT5 DNA binding in the
absence of PRL (Fig. 8A, lanes 2 and 8). An antibody that recognizes
only STAT5, which is tyrosine phosphorylated on tyrosine 700, was used
to examine STAT5a tyrosine phosphorylation using direct Western
blotting (A. V. Kazansky, E. B. Kabotyanski, J. Yeh, S. L.
Wyszomierski, and J. M. Rosen, submitted). The decrease in STAT5a DNA
binding caused by ER was reflected by a parallel decrease in STAT5a
tyrosine phosphorylation (Fig. 8B; compare lanes 4 and 6 to lanes 3 and
5). ER did not affect the level of STAT5a protein (Fig. 8C). Finally,
the effect of ER on STAT5 DNA binding was examined at different times
after PRL treatment to determine whether ER affected the rate of STAT5
inactivation. STAT5a DNA binding after PRL stimulation decreased with
similar kinetics in cells cotransfected with STAT5a and ER and cells
transfected with STAT5a alone (Fig. 8D). Therefore, the GR-dependent
enhancement of STAT5 tyrosine phosphorylation is clearly not a general
effect of all steroid hormone receptors. GR enhanced STAT5
phosphorylation and DNA binding by decreasing the rate of STAT5
dephosphorylation, whereas ER decreased the initial level of
STAT5 tyrosine phosphorylation but had no detectable effect on the
kinetics of dephosphorylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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STAT5 and GR interaction and nuclear translocation as a consequence of a single ligand-induced activation of a respective signaling pathway has now been demonstrated both by IIF in this study and quantitative Western blots using nuclear and cytoplasmic fractions from HC11 cells (30). In the latter study, these investigators found 1.5 times more GR in the nucleus of HC11 cells after PRL treatment compared with no hormone treatment. A similar increase in nuclear STAT5 was observed when only glucocorticoid treatment was performed. Thus, it is conceivable that, under some circumstances, inactive STAT5 and GR may be in the nucleus complexed with their activated partner. The physiological significance of this observation remains to be determined.
Protein tyrosine phosphatases located in the nucleus have been
implicated in STAT inactivation (36). Our data suggest that when STAT5
is complexed with GR, protein-protein interactions decrease the
affinity of STAT5 for a deactivating phosphatase. This could be through
a mechanism involving steric hindrance, particularly if GR and the
phosphatase interact with the same region or nearby regions of STAT5.
Because GR can enhance the DNA binding activity of STAT5a2, it appears
that the carboxy terminus of the STAT5 is not necessary for this
effect. Although the carboxy terminus of STAT5 has been suggested as a
potential STAT5-phosphatase interaction site (29), the amino-terminal
sequences of STAT proteins may also be important for phosphatase
interactions (37). The amino terminus is a region of high sequence
homology between the STAT proteins. Eight 
-helical regions are
predicted, based on amino acid sequence, in the amino termini of all
STATs. The crystal structure of STAT4 revealed that these helices form
a hook-shaped structure that potentially can mediate a variety of
protein-protein interactions (38). When STAT1 is truncated by 61 amino
acids at the amino terminus, tyrosine dephosphorylation is inhibited.
This results in a high basal level of phosphorylation and prolonged
activation after stimulation. Arg 31 and Glu 39 have been shown to be
particularly important and are conserved in all STAT proteins including
STAT5a and STAT5b (37). In addition, a region slightly upstream of the
STAT5 DNA-binding domain (DBD) has been implicated in STAT5
dephosphorylation. The double mutation of amino acid 299 from histidine
to arginine and amino acid 711 from serine to phenylalanine resulted in
prolonged tyrosine phosphorylation of STAT5 after cytokine stimulation
(39). Once the regions of STAT5 that interact with GR are more
precisely delineated and the specific nuclear tyrosine phosphatase is
defined, it should be feasible to test directly this hypothesis.
The data presented in this study are consistent with the hypothesis that GR decreases the affinity of STAT5 for an inactivating phosphatase, which then leads to enhancement and prolongation of STAT5 DNA binding. Alternatively, GR interaction could increase the affinity of STAT5 for DNA, thereby making it less accessible to an inactivating phosphatase resulting in prolonged tyrosine phosphorylation. The latter hypothesis appears less likely, because these experiments are performed in cells transfected with STAT5a, GR, and PRL-R but not a target promoter sequence. Both COS and CHO cells would be required to have an accessible endogenous promoter containing a STAT5-binding site, and possibly 1/2 GREs similar to those found in the ß-casein promoter for the second hypothesis to explain the effect. Although protein-protein interactions between STAT5 and GR are well established, the possibilities either that GR is prolonging STAT5 tyrosine phosphorylation by interacting directly with a phosphatase or perhaps acting upstream to increase STAT5 activation via the PRL-R and JAK2 cannot yet be eliminated.
Cotransfection with ER led to a decrease in tyrosine phosphorylation and DNA binding of STAT5 without affecting STAT5 protein levels. The physiological significance and mechanism of ER modulation of STAT 5 are not understood at present, but may be relevant for a number of genes that are regulated by both cytokines and estrogens. The observed decrease in STAT5 tyrosine phosphorylation is probably the result of a protein-protein interaction involving ER because estradiol was not required to observe this effect. A direct or indirect interaction between the ER and STAT5 would be the most plausible explanation, but this remains to be established. ER clearly does not affect the rate of STAT5 dephosphorylation. Therefore, the decrease in phosphorylation and DNA binding is probably the result of an inhibition of STAT5 activation. Thus, ER may decrease the interaction between STAT5 and PRL-R or the interaction between STAT5 and JAK2. Alternatively, if STAT5 and ER interact in a ligand-independent fashion, similar to STAT5 and GR (30), the decrease in STAT5 tyrosine phosphorylation could result from ER sequestration of a subpopulation of STAT5 in the nucleus away from receptor/JAK2 activation at the cell membrane. Although we do not understand how ER affects STAT5, the experiments presented here demonstrate that steroid receptors other than GR impinge on the JAK/STAT signaling pathway. Progesterone is known to inhibit ß-casein expression during pregnancy (40). PR has been found to inhibit STAT5 induction of ß-casein in transiently transfected CHOk1 cells, and there is evidence of an interaction between STAT5 and PR (E. K. Gass and D. P. Edwards, personal communication). Androgen receptor may also enhance STAT5 tyrosine phosphorylation in a manner similar to GR (S. L. Wyszomierski and J. Rosen, unpublished observations).
The same steroid receptor may also exert selective effects on different STAT proteins. For example, it has been reported that GR interacts and transcriptionally synergizes with both STAT5 (2) and STAT3 (41) on STAT-responsive promoters. An interesting difference occurs on the GR-responsive mouse mammary tumor virus (MMTV) promoter, however. Here STAT3 acts a coactivator with GR to enhance transcription at the MMTV promoter (41), while STAT5 inhibits the MMTV promoter, presumably by sequestering GR in a complex that is incapable of transactivation at the MMTV promoter (2). Further analysis is likely to reveal a plethora of steroid receptor-STAT interactions, each with specific effects and implications based on the proteins involved, the cell type, and the promoter.
While glucocorticoids are known to be essential lactogenic hormones, the mechanisms by which they regulate milk protein gene expression have not been completely defined. Both direct and indirect mechanisms appear to be responsible for steroid hormone regulation of ß-casein gene expression. PRL and HC have been demonstrated to act by kinetically distinct mechanisms in mammary epithelial cells (34). Pretreatment with glucocorticoids is essential for PRL induction of ß-casein gene transcription. This effect is gradually increased with longer glucocorticoid pretreatments, is rapidly reversed when glucocorticoids are withdrawn, and requires ongoing protein synthesis (34, 42). Glucocorticoids may also modulate ß-casein gene transcription through alterations in the levels of different C/EBPß (CCAAT-enhancer binding protein ß) isoforms in an indirect manner requiring new protein synthesis (43). In contrast to this indirect effect, the rapid transcriptional synergy seen by STAT5 and GR is a direct effect on ß-casein gene transcription.
The GR-enhanced EMSA complex detected in these experiments is not likely to contain stably associated GR, despite the presence of 1/2 GREs in the oligonucleotide used for EMSA. A slower mobility EMSA complex was not detected after expression of GR and STAT5 as compared with STAT 5 alone; antibodies to GR had no effect on the mobility of the EMSA complex; and a consensus GRE did not compete in oligonucleotide competition experiments (data not shown). This is consistent with results published by Cella et. al. (30), who were also unable to detect STAT5 and GR in a stable DNA-bound complex on the ß-casein promoter by EMSA. However, by incubating extracts with a ß-casein oligonucleotide followed by immunoprecipitation with antibody to GR, both GR and STAT5 have been detected in a DNA-bound complex on the ß-casein promoter. The formation of this complex was dependent on the presence of an intact GAS site (30).
It is still somewhat controversial whether or not GR binding to the ß-casein promoter is necessary for the observed transcriptional synergy between STAT5 and GR. There are no palindromic GREs in the ß-casein promoter. However, several 1/2 GREs have been mapped in the promoter by in vitro DNAseI footprinting with purified GR (33). The GR DBD alone is capable of binding to some of these 1/2 GREs as a monomer. Mutation of several of the 1/2 GREs individually and in combination abolishes STAT5/GR transcriptional synergy in COS cells. Mutation of the three 1/2 GREs found between -180 and -61 of the ß-casein promoter strongly reduced the synergistic effects of PRL and HC seen in HC11 cells. However, when GR mutants containing either mutated DBDs or the DBD of ER were cotransfected with STAT5 into COS-7 cells, these mutated receptors were still capable of transcriptional synergy with STAT5 (31). The level of synergy was, however, decreased compared with wild-type GR. These results suggest that GR binding to the 1/2 GREs in the ß-casein promoter enhances, but is not absolutely required for, STAT5/GR transcriptional synergy. Of interest in this regard is the recent observation that only a subset of GR functions in vivo were affected by the lack of GR DNA binding in mice where wild-type GR was replaced with DNA binding-defective GR by gene targeting (44). It will be of interest to analyze mammary gland development and functional differentiation in these mutant mice.
While the GR-dependent enhancement of STAT5 phosphorylation and DNA binding is most likely a result of protein-protein interaction between STAT5 and GR, and does not require binding to the ß-casein promoter, binding of both proteins to the ß-casein promoter may amplify in vivo the protective effect of GR on STAT5 phosphorylation seen in these in vitro experiments. Because DNA binding is not needed for GR to enhance STAT5 phosphorylation, this protein-protein interaction has the potential to effect the transcription of any gene induced by STAT5. It could have a greater impact, however, on promoters that are capable of binding both STAT5 and GR, such as the milk protein and acute phase gene promoters.
In the ß-casein gene STAT5, GR, and C/EBPß all participate in transcriptional activation as part of a composite response element (CoRE) (11, 12, 32, 43, 45, 46). In the presence of lactogenic hormones, transcriptional synergy is conferred by a pleiotropic mechanism involving cooperation of the transactivation domains of STAT5 and GR (2, 31) and enhancement of STAT5 DNA binding by GR. GR has also been shown to interact directly with C/EBPß (47), so protein-protein interactions are likely to stabilize the binding of each individual transcription factor to its response element, thereby creating a stable activation complex. STAT5, GR, and C/EBPß have all been shown to interact with p300/CBP (48, 49, 50). Recruitment of coactivators and cointegrators like p300 or CBP to the promoter is likely to be a critical component of transcriptional synergy. Interactions between different classes of transcription factors at CoREs result in highly specific regulation of gene expression (Ref. 51) and references therein), as exemplified by the transcriptional synergy exhibited by GR and STAT5 at the ß-casein CoRE. The observation that GR enhances STAT5 activation by prolonging its tyrosine phosphorylation is an interesting example of how transcription factors in CoREs may be able to modulate each other’s activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture and Transfections
DMEM, trypsin-EDTA, donor horse serum, and glutamine were
purchased from JRH Biosciences (Lenexa, KY). DMEM/F12 and phenol
red-free DMEM were purchased from GIBCO-BRL (Gaithersburg, MD). FBS was
purchased from JRH Biosciences and Summit Biotechnologies (Fort
Collins, CO). Gentamicin, insulin, apo-transferrin, HC, and
ß-estradiol were purchased from Sigma (St. Louis, MO). Ovine PRL (lot
AFP-10677C) was kindly provided by the National Hormone and Pituitary
Program (Bethesda, MD). COS-1 cells and CHOk1 cells were obtained from
the ATCC (Manassas, VA). COS-1 cells were routinely passaged in DMEM +
10% FBS in the presence of gentamicin. COS-1 cell transfections were
performed 1 day after passaging the cells using a lipofectamine
(GIBCO-BRL) protocol. DNA (10–15 µg) and 20 µl of lipofectamine
were used per 100-mm plate. Transfections were performed according to
the manufacturer’s instructions. After transfection, cells were
maintained in DMEM + 10% charcoal-stripped horse serum with gentamicin
and pretreated with 5 µg/ml insulin for 24–48 h. The charcoal
treatment was performed to remove endogenous steroids from the serum.
Treatment with HC (1 µg/ml), ß-estradiol (1 x
10-6 M) and/or ovine PRL (1 µg/ml) was
performed as indicated. CHOk1 cells were routinely passaged in McCoy’s
5a media + 10% FBS in the presence of gentamicin. Transfection of
CHOk1 cells was performed similarly to the COS cells, but serum-free
DMEM/F12 supplemented with 5 µg/ml insulin and 10 µg/ml
apo-transferrin was used throughout the experiment. These serum-free
conditions for CHOk1 cells represent a minor modification to a
previously reported serum-free media for CHOk1 cells (53).
Lipofectamine (10 µl per plate) was used. For COS-1 transfections
done to compare the effects of estrogen receptor to the effects of GR,
Superfect reagent (Qiagen) was used for transfection according to the
manufacturer’s instructions. DMEM without phenol red was used instead
of regular DMEM throughout these experiments.
IIF
Cells were cultured and transfected on glass coverslips, coated
with poly-D-lysine (1 mg/ml, mol wt 70,000–150,000,
Sigma). The coverslips were placed on ice, rinsed two times with
ice-cold PBS, and then fixed with 4% paraformaldehyde in PEM buffer
(80 mM 1,4-piperazinediethane sulfonic acid, 1
mM EGTA, 1 mM MgCl2, pH 6.9) for 30
min. After fixing, all incubations and washes were performed at room
temperature. The coverslips were washed three times with PEM and
incubated with PEM + 1 mg/ml NaBH4 two times for 5 min
followed by washing three times with PEM. Cells were permeablized with
0.5% Triton X-100 in PEM for 10 min. The coverslips were washed three
times with PEM and once with TBST (100 mM Tris, pH 7.4, 150
mM NaCl, 0.1% Tween-20). Before immunostaining, the
coverslips were blocked for 1 h with TBST + 5% nonfat dry milk
(NFDM) (Carnation, Glendale, CA). The coverslips were incubated with
both primary antibodies diluted in TBST + 5% NFDM for 1 h.
Anti-STAT5a C-terminal antibody was used at a 1:200 dilution. BuGR2, a
monoclonal antibody to GR, was used at a 1:500 dilution. After washing
five times with TBST, coverslips were incubated for 30 min in the dark
with both secondary antibodies, antirabbit IgG conjugated with Texas
Red (Southern Biotechnology Associates, Inc., Birmingham, AL),
and antimouse IgG conjugated with FITC (Pierce, Rockford, IL). Both
secondary antibodies were diluted 1:1,000 in TBST + 5% NFDM. The
coverslips were then washed five times with TBST and mounted using
Vectashield mounting media containing 4',6-diamidino-2-phenylindole
hydrochloride (Vector, Burlingame, CA). Images were obtained by
fluorescent microscopy (Zeiss Axiophot; Carl Zeiss, Thornwood,
NY).
Preparation of WCEs
Cells were washed twice with HBSS or PBS without calcium and
magnesium (PBS) to remove media and serum. HBSS or PBS (750 µl) was
added per 100-mm tissue culture dish. Cells were detached by scraping
and transferred to an Eppendorf tube. Cells were pelleted briefly at
15,000 rpm at 4 C. Cell pellets were routinely frozen in liquid
nitrogen and stored at -70 C before extract preparation. Cell pellets
were resuspended in 2–3 volumes of 400 mM Wu buffer (400
mM NaCl, 10 mM HEPES, pH 7.4, 1.5
mM MgCl2, 0.1 mM EGTA, 5%
glycerol, 1 mM dithiothreitol) supplemented with 2 µg/ml
aprotinin, 2 µg/ml benzamidine, 2 µg/ml antipain, 2 µg/ml soybean
trypsin inhibitor, 1.5 µg/ml leupeptin, 1 mM sodium
orthovanadate, and 1 mM sodium molybdate by repeated
pipetting. Most inhibitors were purchased from Sigma. Antipain was
purchased from Boerhinger Mannheim (Indianapolis, IN). Resuspended cell
pellets were incubated on ice for 10–15 min. Mixing by pipetting was
repeated once during the incubation. Extracts were centrifuged at 15000
rpm at 4 C for 10 min to remove cellular debris. Protein levels were
determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA).
All extracts were aliquoted, frozen in liquid nitrogen, and stored at
-70 C. Each aliquot was thawed only once.
EMSA
An oligonucleotide encompassing the ß-casein GAS site and
flanking 1/2 GREs was used for EMSA. The sequence of the coding strand
was as follows: TAATCATGTGGACTTCTTGGAATTAAGGGACTTTT. The sequence for
the coding strand of the oligonucleotide with mutated 1/2 GREs was as
follows: TAATCAAGCTTACTTCTTGGAATTAACAGACTTTT. Oligonucleotides were
designed with 4-bp overhangs and were labeled by filling in the
overhangs with [32P]deoxynucleoside triphosphates (New
England Nuclear LifeScience Products, Boston, MA). Labeled probe was
separated from unincorporated nucleotides using either G-50 spin
columns (Boerhinger Mannheim) or P-6 Micro Bio-Spin columns (Bio-Rad)
according to the manufacturers’ instructions. WCEs were diluted 1:4
with no-salt Wu buffer supplemented with inhibitors (same as above but
without NaCl) to adjust the salt concentration to 100 mM.
Five to 10 µg of total protein were used per reaction. Wu buffer (100
mM) supplemented with inhibitors (same as above with 100
mM NaCl instead of 400 mM) was added so the
total volume was 10 µl. Poly (dI)-poly (dC) (Pharmacia, Piscataway,
NJ) was added (2 µg/reaction). Samples were incubated on ice for 30
min. When competitor oligonucleotides or antibodies were used, they
were included with the extracts during this incubation. Competitors
were added to specific concentrations as indicated. For all antibodies,
1 µl/reaction was used. Five microliters of binding mix [2.5 mg/ml
BSA, 4% Ficoll 400, 10% glycerol, 50 µg/ml p (dN) 5
(Pharmacia)] containing approximately 100 ng labeled probe were added
per reaction. Reaction samples were incubated on ice for 15 min to
allow binding to occur. Reactions were resolved on 5% polyacrylamide
(38:2 acrylamide-bis-acrylamide ratio) gels containing 0.25x TBE and
2.5% glycerol run at 250–275 V at 4 C. EMSAs were quantitated using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Statistical
significance was determined using a two-way, independent t
test.
Immunoprecipitation and Western Blots
Protein A-trysacryl (Pierce) was prepared by washing three times
with RIPA buffer (50 mM Tris, pH 7.4, 150 mM
NaCl, 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate) and
resuspended in RIPA supplemented with inhibitors (1 mM
dithiothreitol, 2 µg/ml aprotinin, 2 µg/ml benzamidine, 2 µg/ml
antipain, 2 µg/ml soybean trypsin inhibitor, 1.5 µg/ml leupeptin, 1
mM sodium orthovanadate, and 1 mM sodium
molybdate) to twice the original volume. Total protein (400 µg per
sample) was used. The total volume was brought to 350–400 µl with
RIPA supplemented with inhibitors. Extracts were precleared by
incubation with 40 µl protein A-trysacryl for 30 min at 4 C with
rocking. STAT5 N-terminal antibody (Santa Cruz STAT5 N-20, Santa Cruz,
CA) was used at a 1:100 dilution. Antibody was incubated for 3 h
with the extracts at 4 C with rocking. Protein A-trysacryl (60 µl)
was then added, and the samples were incubated overnight at 4 C with
rocking. The resin was washed three times with RIPA buffer supplemented
with inhibitors, and bound proteins were eluted by boiling in SDS
sample buffer for 10 min. Proteins were separated by standard SDS-PAGE
techniques utilizing 3% stacking gels and 7.5% running gels. They
were transferred to Immobilon-P polyvinylidene fluoride
membranes (Millipore, Bedford, MA) overnight at 90 mA. Western blots
were done using standard protocols (43) with STAT5a affinity-purified
antibody (15) at a 1:5,000 dilution, STAT5Y700P affinity-purified
antibody at a 1:400 dilution, or PY20 (Transduction Laboratories,
Lexington, KY) at a 1:500 dilution. STAT5Y700P is an affinity-purified
rabbit polyclonal antibody that is specific for STAT5 phosphorylated on
tyrosine 700. It does not recognize STAT5 that is not phosphorylated on
this residue (A. V. Kazansky, E. B. Kabotyanski, J. Yeh, S. L.
Wyszomierski, and J. M. Rosen, submitted). For phosphotyrosine blots,
modified TBST (10 mM Tris, pH 7.5, 100 mM NaCl,
0.1% Tween 20) + 1% BSA was used for blocking and incubation with the
primary antibody. Biotinylated goat antirabbit IgG, biotinylated goat
antimouse IgG, and streptavidin-horseradish peroxidase were purchased
from Calbiochem (La Jolla, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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These studies were supported by NIH Grant CA-16303 from the NIH. S.L.W. was supported by a breast cancer training grant from the Department of Defense (DAMD 17–94-J-4204).
Received for publication July 24, 1998. Revision received October 2, 1998. Accepted for publication October 20, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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