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(Received for publication, June 23, 1995) From the
Binding sites for the tissue-specific transcription factor,
Pit-1, are required for basal and hormonally induced prolactin gene
transcription. Although Pit-1 is phosphorylated in response to several
signaling pathways, the mechanism by which Pit-1 contributes to
hormonal induction of gene transcription has not been defined. Recent
reports suggest that phosphorylation of Pit-1 may not be required for
hormonal regulation of the prolactin promoter. Analysis of the
contribution of individual Pit-1 binding sites has been complicated due
to the fact that some of the elements appear to be redundant. To better
understand the role of Pit-1 sites in mediating hormonal regulation of
the prolactin gene, we have performed enhancer tests using the three
most proximal Pit-1 binding sites of the rat prolactin gene which are
designated the 1P, 2P, and 3P sites. The results demonstrate that
multimers of the 3P Pit-1 binding site are much more responsive to
several hormonal and intracellular signaling pathways than multimers of
the 1P or 2P sites. The 3P DNA element was found to contain a consensus
binding site for the Ets family of proteins. Mutation of the Ets
binding site greatly decreased the ability of epidermal growth factor,
phorbol esters, Ras, or the Raf kinase to induce reporter gene
activity. Mutation of the Ets site had little effect on basal enhancer
activity. In contrast, mutation of the consensus Pit-1 binding site in
the 3P element essentially abolished all basal enhancer activity.
Overexpression of Ets-1 in GH
The transcription of the prolactin gene is modulated by a number
of hormones which bind to plasma membrane receptors. Hormones which
regulate the transcription of the prolactin gene include
dopamine(1) , epidermal growth factor(2, 3) ,
and thyrotropin releasing hormone(4, 5) . The
transcriptional effects of these hormones likely involves the
activation of protein kinases leading to the phosphorylation of
specific transcription factors. It has been demonstrated that
activation of the cAMP-dependent protein kinase(6) , the
Ca In the present study we have
examined the ability of individual Pit-1 binding sites to permit
responses to hormones and activators of specific signal transduction
pathways. Previous studies have suggested that the 5`-flanking region
of the rat prolactin gene contains multiple, redundant DNA elements
which mediate responses to TRH(11) . This functional redundancy
has complicated analysis of the role of specific DNA elements in
mediating hormonal responsiveness. Because of this problem, we have
used an enhancer test to compare the ability of the three most proximal
Pit-1 binding sites of the prolactin gene to respond to hormones,
intracellular second messengers and activated components of signal
transduction pathways. We find that one site, the 3P site, is
particularly responsive to several signal transduction pathways
including activation of the MAPK cascade. We have further examined the
DNA sequences of the 3P DNA element which are important for
transcriptional regulation. These studies suggest that the 3P element
contains a binding site for a member of the Ets family of transcription
factors in addition to a Pit-1 binding site. The Ets site in the 3P DNA
element is crucial for transcriptional responses to hormones and second
messengers.
Figure 1:
Enhancer
activity in rat GH
Presumably, the ability of the 3P
multimer to facilitate transcriptional responses is dependent on
binding of Pit-1 to these elements. On the other hand, although Pit-1
is known to bind to both the 1P and 3P elements, the 3P element
multimer was much more responsive to activation of the MAPK pathway.
Pit-1 expression is restricted to somatotrophs, lactotophs, and a
subset of thyrotroph cells of the anterior
pituitary(42, 43, 44) . To directly assess
the role in Pit-1 in mediating transcriptional responses, we
transfected the same reporter genes in the presence or absence of a
Pit-1 expression vector into Rat-1 cells which do not contain
endogenous Pit-1 (Fig. 2). In the absence of the Pit-1
expression vector, the proximal prolactin promoter, the 1P multimer
construct and the 2P multimer construct were all essentially inactive
in Rat-1 cells. The 3P multimer did support a very low level of basal
expression. Transfection of the Pit-1 expression vector substantially
activated the proximal prolactin reporter gene (23-fold) as well as the
constructs containing the 1P (49-fold) or the 3P multimers (43-fold).
The 2P multimer did not respond to the Pit-1 expression vector in Rat-1
cells. Transfection of the Pit-1 expression vector enabled the 1P
multimer to support a modest response to cAMP as has been reported
previously(18) . Although both the proximal prolactin promoter
and the 3P multimer supported responses to phorbol esters in GH
Figure 2:
Enhancer activity of Pit-1 binding site
multimers in Rat-1 fibroblast cells. The reporter genes described in Fig. 1were transfected by electroporation into Rat-1 cells along
with an expression vector for globin (panel A) or for Pit-1 (panel B). Each transfection was divided evenly into three
plates. The next day the cells were untreated (Control) or
treated with 0.5 mM CPT-cAMP or 100 nM PMA for 6
hours prior to collection and assaying for luciferase activity. Values
are the average ± standard error of three independent
transfections.
Figure 3:
Binding of purified Pit-1 to the 1P and 3P
elements. The nucleotide sequences of the 1P and 3P DNA elements are
indicated (A). Boxes indicate regions previously
demonstrated to be protected by GH
We
also explored the binding of endogenous factors from GH
Figure 4:
Binding of GH
Figure 5:
Mutational analysis of the prolactin 3P
site. The nucleotide sequence of the wild type and mutant 3P elements
are indicated (A). For the wild type sequence, two consensus
binding sites for Pit-1 and a binding site for members of the Ets
family of transcription factors are enclosed with boxes. The
binding of purified Pit-1 to wild type and mutant 3P site DNA probes
was analyzed by incubation of radiolabeled DNA probes with increasing
concentrations of Pit-1 and resolution of free probe and complexes by
non-denaturing polyacrylamide gel electrophoresis. Binding was
quantitated by PhosphorImager analysis and affinities were calculated.
The binding data are normalized to wild type DNA element and represent
the average ± standard error of three independent experiments (B). To determine basal enhancer activity (C) or
inducible enhancer activity (D), reporter genes carrying four
copies of wild type or mutant 3P elements upstream of a minimal
promoter were transfected into GH
Figure 6:
Binding of Pit-1 and the DNA binding
domain of the Ets factor ER81 to wild type and mutant 3P DNA elements.
The wild type, mut1, or mut4 3P DNA elements (sequence of probes
indicated in Fig. 5) were incubated with Pit-1 or the DNA
binding domain of ER81 as indicated, and complexes were resolved by
non-denaturing polyacrylamide gel electrophoresis.
Poly(histidine)-tagged ER81 was expressed in E. coli and purified by nickel chelate
chromatography.
To provide a functional test for the interaction of Ets factors with
the 3P element, an expression vector for human c-Ets-1 was
cotransfected with reporter genes containing multimers of the 3P site (Fig. 7). Overexpression of Ets-1 stimulated expression of the
reporter gene containing four copies of the wild type 3P enhancer. As
shown earlier, the wild type 3P element can support a response to Ras.
The ability of a reporter gene to respond to Ets-1 or Ras was
essentially eliminated by mutation of the putative Ets binding site
(mut1). These studies demonstrate that the putative Ets factor binding
site of the 3P element is essential for mediating responses to both
Ets-1 and Ras.
Figure 7:
Overexpression of Ets-1 in GH
Figure 8:
A GAL4-Elk1 fusion protein responds to
multiple signal transduction pathways in GH
These studies have shown that multiple copies of the 3P DNA
element of the rat prolactin gene can function as an inducible enhancer
mediating transcriptional activation in response to TRH, PMA, and EGF
as well as activated forms of Ras and the Raf kinase. The ability of
the 3P element to mediate these responses has been found to require
intact binding sites for two different transcription factors, Pit-1 and
an Ets factor. By itself, the Pit-1 binding site of the 3P element
appears to be sufficient for basal enhancer activity. Although the
Pit-1 binding site is necessary to permit activation in response to PMA
or the Raf kinase, it is not sufficient for this response. The Ets
factor binding site does not appear to contribute to basal enhancer
activity but is necessary for responses to PMA or raf. Thus, the Ets
and Pit-1 binding sites functionally cooperate to permit hormonal
responsiveness. A number of previous studies led to the view that
Pit-1 is important for mediating hormonal responsiveness of the
prolactin gene. In several studies, Pit-1 binding sites were shown to
be important for mediating hormonal regulation of the prolactin
promoter(13, 14, 16) . The observation that
Pit-1 is phosphorylated in vivo after treatment with cAMP,
PMA, or TRH (17, 20) is also consistent with a role
for Pit-1 in mediating transcriptional responses to these agents. This
might suggest a simple model in which phosphorylation of Pit-1
modulates its transcriptional activity. However, several recent studies
have reached the surprising conclusion that although Pit-1 is required
for hormonal regulation of the prolactin gene, phosphorylation of Pit-1
is apparently not required(18, 19, 20) . What
then is the role of Pit-1 in mediating hormonal regulation of
transcription? It is clear that Pit-1 is essential for tissue-specific
expression of the prolactin
gene(10, 42, 50, 51, 52) .
As has been previously suggested, the participation of Pit-1 in
hormonal regulation of the prolactin gene may provide a mechanism which
permits the linking of ubiquitous signal transduction pathways to
cell-specific transcriptional responses(21, 36) . The
present studies suggest that cooperation between Pit-1 and an Ets
factor likely provides a mechanism permitting a tissue-specific
transcriptional response to activation of the MAPK pathway. Ets
factors appear to frequently act in concert with other transcription
factors and a subset of Ets factors mediate transcriptional responses
to the MAPK pathway. There are now numerous examples in which an Ets
factor appears to cooperate with another transcription factor to
mediate transcriptional responses. For instance, the Ets factor Elk1
interacts with the serum response factor as a crucial step in mediating
the ability of the fos promoter to respond to growth
factors(49, 53, 54) . Similarly, other
members of the Ets family have also been found to functionally interact
with other transcription factors, often to permit synergistic
activation of specific
genes(31, 55, 56, 57, 58, 59, 60, 61, 62) In
view of the ability of the prolactin 3P element to permit responses to
activators of the MAPK cascade, it is particularly interesting that
Elk1 contains several MAPK phosphorylation sites. The use of fusions in
which the carboxyl-terminal domain of Elk1 was linked to a heterologous
DNA binding domain have demonstrated that the Elk1 contains a
transcriptional activation domain which is regulated by MAPK
phosphorylation(49) . SAP1 and Net which are related to Elk1
have also been shown to be regulated by Ras and the MAPK
pathway(63, 64) . A combination of genetic and
biochemical studies have clearly demonstrated that the Drosophila Ets factors, Yan and Pointed, are regulated by the Ras/MAPK
pathway(65, 66) . Thus, it is well established that
Ets factors can participate in mediating MAPK-induced transcriptional
activation. Multimers of the 3P site can support transcriptional
responses to a number of diverse signaling pathways including TRH, EGF,
phorbol esters, cAMP, Ras and Raf. It is possible that activation of
the MAPK pathway leading to phosphorylation of an Ets factor could be
involved in all of these responses. The finding that the Ets site is
required for all of the responses is consistent with this model.
However, is there evidence for activation of MAPK by all of these
pathways? Of course, EGF, Ras, and Raf would be expected to activate
MAPK. Recent studies have shown that TRH treatment activates MAPK in
GH In a number of respects
the present findings are similar to a recent report from Bradford et al.(73) which examined the interaction of c-Ets-1
and Pit-1 in mediating the ability of Ras to activate the prolactin
promoter. Bradford et al. focused their attention on a
different Pit-1 binding site, the 4P site. They demonstrated that there
is an Ets factor binding adjacent to the 4P Pit-1 binding site. They
found that overexpression of both Pit-1 and Ets-1 enhanced
responsiveness of the prolactin promoter to Ras. Our studies
demonstrate that the 3P site also contains an Ets factor binding site
which is adjacent to a Pit-1 binding site. Taken together, the two
studies provide strong evidence that Pit-1 can functionally synergize
with Ets factors and that this interaction appears to be important for
mediating transcriptional responses to the MAPK pathway. As noted
above, the identity of the endogenous Ets factor which binds to the 3P
element has not yet been determined. Further understanding of
transcriptional regulation of the prolactin gene will require
identification of the endogenous proteins which interact with these
sequences and characterization of their phosphorylation in response to
hormonal stimulation.
Volume 270,
Number 36,
Issue of September 08, pp. 20930-20936, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
pituitary cells enhanced both
basal and Ras induced activity from the 3P enhancer. These data
describe a composite element in the prolactin gene containing binding
sites for two different factors and the studies suggest a mechanism by
which Ets proteins and Pit-1 functionally cooperate to permit
transcriptional regulation by different signaling pathways.
/calmodulin-dependent protein kinase type
II(7) , or the MAPK (
)cascade (8) is
sufficient to stimulate transcription of the prolactin gene. Despite
rather extensive studies of the prolactin promoter, the mechanisms
which permit transcriptional responses to different hormones and signal
transduction pathways have not been clearly defined. It is clear that
the prolactin promoter contains multiple binding sites for the
tissue-specific transcription factor Pit-1(9, 10) ,
and there is evidence that Pit-1 binding sites may contribute to both
basal and hormonally regulated
transcription(11, 12, 13, 14, 15, 16) .
The observation that treatment of GH cells with cAMP or
phorbol esters stimulates phosphorylation of Pit-1 (17) is
consistent with a role for Pit-1 in mediating hormonal regulation of
transcription. However, recent studies have shown that phosphorylation
of Pit-1 may not be necessary for hormonal induction of prolactin gene
transcription (18, 19, 20) . This led to the
suggestion that other factors which interact with Pit-1 binding sites
may mediate transcriptional regulation of the prolactin gene or that
co-activators may interact with Pit-1 in a regulated
fashion(21) . There is evidence that cooperative interactions
between Pit-1 and other transcription factors may be crucial for some
transcriptional
responses(12, 15, 22, 23) . It has
also been shown that factors other than Pit-1 can interact with Pit-1
binding sites. For instance, Oct-1(24) , Zn-15(25) ,
and TEF (26) can all interact with Pit-1 binding sites and
activate transcription through these sites. However, it is not clear if
any of these other factors play a role in mediating hormonally
regulated transcriptional activation.
Transfection of GH
GH and Rat-1
Cells cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 15% equine serum and
2.5% fetal bovine serum. Rat-1 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
calf serum. Reporter genes containing 0.6 kilobase pairs of 5`-flanking
region of the rat prolactin gene fused to the firefly luciferase coding
sequence (11) and five copies of a GAL4 binding site upstream
of the E1b TATA box linked to luciferase (27) have been
described previously. To prepare a luciferase reporter gene (28) containing multiple copies of the prolactin gene proximal
Pit-1 binding sites, the rat prolactin 5`-flanking region was truncated
at position -33 and a non-palindromic AvaI site was
placed at the upstream termini. Multiple, tandem copies of Pit-1
binding sites with AvaI cohesive termini were ligated upstream
of the minimal prolactin promoter and luciferase gene. GH and Rat-1 cells were transfected by electroporation with
10-15 µg of luciferase reporter genes using conditions
described previously(29, 30) . In some experiments the
cells were transfected with expression vectors encoding globin,
Pit-1(11) , Raf-BXB, a constitutively active form of the Raf
kinase(31) , human Ets-1(32) , or a H-ras valine 12
mutant(33) . After electroporation, the cells were divided
equally into 60-mm tissue culture plates. At 18 h after transfection,
some of the cultures were treated with 0.5 mM 8-(4-chlorophenylthio)cAMP, 100 nM TRH, 10 nM murine EGF, or 100 nM PMA for an additional 6 h. The
cultured cells were collected and assayed for luciferase activity as
described (34) .Mobility Shift Assays for Analysis of Protein-DNA
Interactions
Recombinant Pit-1 containing a six histidine
amino-terminal tag was expressed in Pichia pastoris and
purified on nickel chelate agarose as recommended by the manufacturer
(Invitrogen). Recombinant histidine-tagged ER81 was produced in Escherichia coli and purified by nickel chelate
chromatography. Nuclear extracts were prepared from GH cells as described previously(30) . Binding reactions
contained 10,000 counts/min of P-labeled DNA probe,
varying amounts of Pit-1, 2 µg of sheared salmon sperm DNA, 20
µg of bovine serum albumin, 10 mM Tris, pH 7.5, 5%
glycerol, 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol in a total volume of 25 µl. In some reactions 1
µl of preimmune serum or antiserum to Pit-1 was included. The
antiserum to Pit-1 has been described previously (18) (20).
Reactions were incubated for 30 min at room temperature and then
analyzed on non-denaturing, 4% polyacrylamide gels. Quantitation of
mobility shift gels was performed using a Molecular Dynamics
PhosphorImager Si and Imagequant Software. Relative dissociation
constants and Hill cooperativity coefficients were determined by
non-linear curvefitting of the Hill equation to the binding data.
Enhancer Activity of Multimers of Pit-1 Binding
Sites
In an effort to provide a relatively simple test system
for analysis of possible mechanisms mediating hormonal responsiveness
of the prolactin promoter, we tested the ability of individual Pit-1
binding sites to function as hormone-responsive enhancers. This
approach is based on the observation that there appears to be
functional redundancy in the DNA elements which permit hormonal
regulation of the prolactin promoter (11, 35, 36) and the finding that multimers
of individual Pit-1 binding sites can support transcriptional responses
to some hormones or second messengers(14, 18) . The
proximal region of the prolactin gene is sufficient to mediate
transcriptional responses to several different
hormones(2, 35, 37, 38) , and this
region contains four Pit-1 binding sites which are designated P1
through P4(9, 10) . We have focused our attention on
the three most proximal Pit-1 binding sites (P1, P2, and P3) as we
previously found that mutation of the P4 Pit-1 binding site had little
effect on hormonal responsiveness(11) . We prepared
multimerized, direct repeats of the P1, P2, and P3 Pit-1 binding sites
which were placed upstream of the minimal TATA box of the prolactin
gene and the luciferase coding sequence. The reporter genes containing
the multimers were then tested for hormonal regulation by transfection
of the GH, rat pituitary cell line (Fig. 1). The
response of the multimers was compared to a reporter gene containing
600 base pairs of the proximal region and promoter of the rat prolactin
gene (0.6PRL). A reporter gene containing the thymidine kinase (TK) promoter was included as a control for nonspecific
effects of agonist treatments. The minimal prolactin promoter (TATA box
alone) was essentially inactive. All three Pit-1 binding site multimers
enhanced basal expression of the reporter gene. The 1P site is the
highest affinity Pit-1 binding site in the proximal prolactin
promoter(9) , and the 1P multimers were the strongest basal
enhancer. Multimers of the 1P site also supported a modest response to
cAMP as reported previously(18) . Although it has been reported
that multimers of the 1P site are able to permit a response to
TRH(14) , we could not confirm this observation. This may
reflect differences in culture conditions as the previous studies used
serum-free medium while we used serum-containing medium. Multimers of
the 2P site were found to be a rather weak basal enhancer and did not
appear to permit responses to hormones or second messenger treatments.
Although Pit-1 binding to the 2P site has been
demonstrated(10) , there is also evidence that this DNA element
can interact with a ubiquitous factor (39) and that it plays a
role in repressing the activity of the prolactin promoter in
non-pituitary cell types(40) . Multimers of the 3P site
demonstrated substantial basal enhancer activity and also permitted
responses to cAMP, TRH, and PMA. Indeed, a reporter gene containing
seven copies of the 3P site was substantially more responsive to
phorbol esters than the native prolactin promoter. These findings
demonstrate that multimers of the 3P element are sufficient to confer
responses to at least two different signal transduction pathways and
suggest that this element may contribute to the ability of the
prolactin promoter to respond to several different hormones. We were
particularly interested in the finding that the 3P multimer was more
responsive to TRH than the 1P multimer. TRH has been reported to
activate the MAPK pathway in GH cells(41) , and the
prolactin promoter has been shown to be responsive to activation of
MAPK cascade(8) . Therefore, we tested the ability of the
multimerized DNA elements to mediate responses to activation of the
MAPK pathway (Fig. 1B). For these studies we used
expression vectors encoding activated forms of ras (33) or the
raf kinase (31) which would be expected to stimulate the
activity of the MAPK cascade. As previously reported(8) , the
prolactin promoter is quite responsive to expression vectors for
activated Ras or Raf. While neither the 1P nor 2P multimers permitted a
substantial response to the Ras or Raf expression vectors, the 3P
multimers conferred a vigorous response. The results shown in Fig. 1were obtained using multimers containing seven copies of
the 1P, 2P, and 3P elements. Similar results have been obtained using
multimers containing four copies of the DNA elements (data not shown).
These enhancer tests suggest that the 3P element is particularly
capable of responding to multiple signal transduction pathways
including the MAPK cascade.
pituitary tumor cells of Pit-1 binding
site multimers. Luciferase reporter genes were prepared which contained
a minimal promoter from the prolactin gene and either no additions (TATA) or seven upstream, tandem copies of the 1P, 2P, or 3P
Pit-1 binding sites (7 1P, 7
2P, and 7
3P). Luciferase reporter genes containing the
proximal 0.6 kilobase pairs of the 5`-flanking region of prolactin gene (0.6PRL) or the thymidine kinase promoter (TK) were
also tested. In some experiments the cells were also transfected with
expression vectors for constitutively active forms of Ras or Raf as
indicated. Cells were transfected by electroporation and divided evenly
into four plates. The next day the cells were untreated (Control) or treated with 0.5 mM CPT-cAMP (cAMP), 100
nM TRH, or 100 nM PMA for 6 hours prior to collection
and assaying for luciferase activity. Values are the average ±
standard error of three independent
transfections.
cells, these reporter genes did not respond to phorbol esters in
Rat-1 cells even in the presence of the Pit-1 expression vector. This
difference may indicate that tissue-specific factors other than Pit-1
are required for these regulatory responses. Alternatively, there may
be differences in the signal transduction machinery of GH and Rat-1 cells.
Analysis of Pit-1 Binding to the 1P and 3P
Elements
Multimers of either the 1P or 3P Pit-1 binding site can
provide basal enhancer activity. However, only the 3P multimer can
support substantial transcriptional responses to TRH, phorbol esters,
or activation of the MAPK cascade. It is certainly likely that this
difference in transcriptional regulation mediated by the 1P and 3P
multimers would involve differences in protein binding to these
specific elements. This could reflect quantitative or qualitative
differences in the binding of Pit-1 to these elements. Alternatively,
the differential regulation could involve the binding of factors other
than Pit-1 to the DNA elements. The possibility of qualitative
differences in Pit-1 binding to these two sites is suggested by the
organization of the two DNA elements (Fig. 3A).
Comparison of the 1P and 3P DNA elements to the consensus Pit-1 binding
site, TATTCAT (9, 45, 46) demonstrates that
the 1P site contains two Pit-1 monomer sites arranged in an imperfect
palindrome. The 3P site can be considered to contain a direct repeat of
the monomer binding site. Previous studies have shown that Pit-1
interacts with the 1P element as both a monomer and a dimer with the
dimer forming cooperatively as the concentration of Pit-1 is
increased(47) . A detailed analysis of Pit-1 binding to the 3P
element has not been performed. Therefore, we compared the ability of
purified Pit-1 to bind to the 1P and 3P elements using a mobility shift
assay (Fig. 3, B and C). With the 1P element,
addition of increasing concentrations of Pit-1 resulted in the
formation of two complexes (C1 and C2). Previous studies have shown
that the C1 complex appears to represent the binding of Pit-1 monomers
while the C2 complex represents binding of Pit-1 dimers(47) .
As observed by Ingraham et al.(47) , the formation of
the C2 complex on the 1P element is greatly facilitated over a narrow
range of Pit-1 concentrations consistent with cooperative formation of
Pit-1 dimers. Binding of Pit-1 to the 3P element also demonstrated the
formation of two complexes which were similar in mobility to those
observed with the 1P probe. However, the C1 complex persisted at much
higher concentrations of Pit-1, and there was reduced formation of the
C2 complex at all concentrations of Pit-1. These findings suggest
reduced cooperativity in the binding of Pit-1 to the 3P element.
Quantitative analysis of the mobility shifts shown in Fig. 2, A and B, by PhosphorImager confirmed these
conclusions. The overall affinity of Pit-1 for the 3P element was about
60% of that for the 1P element. Binding to the 1P element occurred with
a Hill coefficient of 1.5 consistent with substantial cooperativity
while a Hill coefficient of 1.1 was obtained for the 3P DNA element.
Similar results have been obtained in several independent binding
experiments. The reduced cooperativity of binding and decreased
apparent formation of Pit-1 dimers at the 3P site raises the
possibility that Pit-1 may interact with this site as a monomer.
nuclear extract from
DNase cleavage. The arrows indicate the presence of consensus
Pit-1 binding sites. For analysis of Pit-1 binding to the 1P (B) or 3P element (C), poly-histidine-tagged Pit-1
was expressed in P. pastoris and purified by nickel
chelate chromatography. DNA probes containing one copy of the proximal
prolactin 1P or 3P Pit-1 binding sites were labeled with P
and incubated with increasing amounts of Pit-1 protein. Bound complexes
were then resolved on non-denaturing polyacrylamide gels. The mobility
of free, uncomplexed DNA probe (free) as well as two different
complexes (C1 and C2) are
indicated.
cells to the 1P and 3P sites. Radiolabeled probes representing
the 1P and 3P sites were tested for their ability to interact with
GH cell nuclear proteins in a gel mobility shift assay (Fig. 4). Multiple protein
DNA complexes were detected with
both probes. Two major complexes observed with the 1P probe were
designated complex-1 (C1) and complex-2 (C2). These
complexes are similar in mobility to the C1 and C2 complexes formed
with purified Pit-1 (data not shown) and likely represent binding of
Pit-1 monomers and dimers, respectively. However, the multiple bands
observed with total nuclear proteins prevent definitive assignments.
The C1 and C2 complexes were also observed with the 3P probe, but the
C2 signal was much weaker. In an effort to enhance detection of factors
other than Pit-1 which bind to the 1P and 3P DNA element, antiserum to
Pit-1 was included in the binding reactions. Formation of
proteinDNA complexes with either the 1P or the 3P probe was
completely disrupted by antiserum to Pit-1. Preimmune serum had little
or no effect on binding. The Pit-1 antiserum had no effect on the
binding of GH nuclear proteins to a cyclic AMP response
element (data not shown), further confirming the specific effect of the
antiserum. Unfortunately, this approach did not permit detection of any
proteinDNA complexes which were resistant to the antiserum. This
finding does provide strong evidence that Pit-1 is a major component of
the endogenous GH factors which bind to both the 1P and 3P
DNA elements. Of course, we cannot rule out the possibility that
factors other than Pit-1 may be included in the endogenous proteins
which bind to the 1P and 3P elements but that these other factors
require the presence of Pit-1 for stable interaction with the 1P and 3P
elements.
nuclear proteins
to the 1P and 3P elements. Radiolabeled DNA probes representing either
the 1P or 3P DNA elements were incubated with increasing concentrations
of GH nuclear extracts in the absence or presence of
antiserum to Pit-1 as indicated. The formation of proteinDNA
complexes was analyzed by non-denaturing polyacrylamide gel
electrophoresis.
Mutational Analysis of the 3P DNA Element
The
preceding experiments provided evidence that multimers of the 3P DNA
element are much more capable of mediating transcriptional responses to
TRH, phorbol esters, or activation of MAPK than are multimers of the 1P
element. Furthermore, Pit-1 binds cooperatively to the 1P element but
with little or no detectable cooperativity at the 3P element. To
further explore the relationship between Pit-1 binding and hormonal
responsiveness of the 3P site, a series of clustered point mutations
were introduced into this DNA element (Fig. 5A). The
binding of Pit-1 to the wild type and mutant DNA elements was then
tested by performing mobility shift analysis with increasing
concentrations of Pit-1 for each DNA and calculation of a relative
affinity from the quantitative titration data (Fig. 5B). Multimers of each mutant DNA were also
prepared and tested for basal and regulated enhancer activity (Fig. 5, C and D). Interestingly, two of the
mutations (mut1 and mut2) had either very little or
no effect on Pit-1 binding but dramatically reduced the ability of
multimers to mediate transcriptional regulation in response to PMA,
EGF, or Raf. One of these mutations (mut1) is completely outside of the
two putative Pit-1 monomer binding sites. We noticed that both mut1 and
mut2 disrupted a DNA sequence, TTCC, which represents a core site for
the Ets family of transcription factors. Another set of mutations (mut4, mut5, and mut6) substantially
decreased Pit-1 binding and reduced basal and regulated enhancer
activity of DNA multimers. All of the mutations which reduced both
Pit-1 binding and basal enhancer activity were found to disrupt DNA
sequences within the downstream Pit-1 monomer binding site (Pit-1
Site-a, indicated in Fig. 5A). One of the
mutations, mut3, disrupts sequences within the center of the upstream
putative Pit-1 binding site (Pit-1 Site-b). As mut3 has very
little effect on either basal or inducible enhancer activity, it seems
likely that Pit-1 does not bind to this site in vivo. The
clustered point mutation analysis of the 3P element provides evidence
that binding of Pit-1 is necessary but not sufficient to permit
inducible transcription. All of the mutations which substantially
reduce Pit-1 binding, also reduce basal and inducible reporter gene
expression. Although mut1 did not disrupt Pit-1 binding, it greatly
reduced the ability of 3P multimers to mediate responses to phorbol
esters, EGF, and the Raf kinase. These findings suggest a possible
model in which the 3P DNA element contains binding sites for Pit-1 and
another factor and both of these binding sites are required for
transcriptional responses to activation of MAPK.
cells. All values are the
average ± standard deviation of three independent transfections
and are normalized to the wild type 3P enhancer reporter
gene.
An Ets Factor Can Interact with the 3P Enhancer
Element
The presence of a putative Ets binding site within the
3P DNA element raised the possibility that this site may serve as an
Ets factor binding site. To test the ability of Ets factors to interact
with the 3P enhancer, the DNA binding domain of an Ets factor, human
ER81(48) , was produced in E. coli and tested for
binding to wild type and mutant 3P DNA elements (Fig. 6). The
results demonstrate that the DNA binding domain of ER81 is able to bind
to the wild type 3P element. This binding appears to be specific as a
3-base pair mutation which disrupts the core Ets 1 binding site (mut1)
greatly reduces binding of ER81. In contrast, a mutation which disrupts
the consensus Pit-1 binding site (mut4) had no effect on the binding of
ER81. In mixing experiments, we found that the presence of Pit-1 did
not enhance the binding of ER81 to the 3P element (data not shown).
cells can increase enhancer activity of a wild type, but not a
mutant 3P DNA element. Luciferase reporter genes containing four copies
of the wild type or the 3P mut1 enhancers were transfected into
GH cells with a cytomegalovirus promoter expression vector
or the same vector driving expression of human Ets1 or constitutively
active Ras as indicated. Values are the average ± standard
deviation of three independent
transfections.
Several Different Signal Transduction Pathways Can
Activate the Elk1 Transcription Factor in GH
The preceding studies have shown that an Ets factor
binding site in the 3P element is essential to permit transcriptional
regulation by several different signal transduction pathways. As the
Ets family member Elk1 can mediate transcriptional activation in
response to growth factors and other activators of the MAPK
pathway(49) , we explored the possibility that TRH, EGF,
phorbol esters, and cAMP might be able to activate Elk1 in GH Cells cells. An expression vector encoding the DNA binding domain of
GAL4 fused to the carboxyl-terminal transcriptional activation domain
of Elk1 was cotransfected with a reporter gene containing five copies
of the GAL4 binding site upstream of a minimal TATA box and the
luciferase coding sequence. The GAL4-Elk1 fusion protein supported
transcriptional responses to TRH, EGF, phorbol esters, and cAMP (Fig. 8). These studies demonstrate that diverse signal
transduction pathways which can mediate transcriptional activation
through the 3P DNA element can also activate a specific member of the
Ets family of transcription factors. Of course, we have not identified
the endogenous Ets factor which binds to the 3P DNA element, and this
study has simply used Elk1 as a model to determine if these signal
pathways can activate an Ets factor. As Elk1 is known to be responsive
to activation of the MAPK pathway(49) , these studies raise the
possibility that the ability of the 3P DNA element to permit
multihormonal regulation may involve convergent regulation of the MAPK
pathway.
cells.
Cytomegalovirus promoter driven expression vectors coding for the DNA
binding domain of GAL4 or the DNA binding domain of GAL4 fused to the
carboxyl-terminal transcriptional activation domain of Elk1 (GAL4-Elk1) were transfected with a luciferase reporter gene
containing five copies of the GAL4 binding site upstream of a minimal
TATA box. 18 h after transfection cultures received no addition
(control), 0.5 mM chlorophenylthio-cAMP (CPT-cAMP),
100 nM PMA, 100 nM TRH, or 10 nM EGF for 6
h. Values are the average ± standard deviation of three
independent transfections.
cells through a mechanism which is at least partially
dependent on protein kinase C(41) . This is consistent with the
observation that protein kinase C can phosphorylate and activate Raf (67, 68) and that TRH treatment leads to activation of
protein kinase C(69, 70) . While it might be
surprising to suggest that cAMP could also activate MAPK, recent
studies provide evidence for tissue-specific differences in the effects
of cAMP on MAPK activity. Although cAMP decreases MAPK activity in many
cells, there is evidence that cAMP can increase MAPK activity in at
least some cells(71, 72) . The present studies have
shown that cAMP as well as EGF, TRH, and phorbol ester treatment of
GH cells can increase the transcription stimulating
activity of a GAL4-Elk1 fusion protein. As GAL4-Elk1 has been shown to
mediate transcriptional responses to MAPK, these results raise the
possibility that cAMP may stimulate MAPK activity in GH cells. Thus it is conceivable that the multihormonal regulation
of the 3P element involves convergent activation of MAPK. Further
studies of cAMP effects on MAPK activity in GH cells will
be required to further address this issue.
)
We thank B. Maurer for aid in preparing the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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