(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 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.
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/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.
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 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.
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 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.
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 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.
We
also explored the binding of endogenous factors from GH 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
protein
DNA 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
protein
DNA 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.
Figure 4:
Binding of GH 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 protein
DNA
complexes was analyzed by non-denaturing polyacrylamide gel
electrophoresis.
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 cells. All values are the
average ± standard deviation of three independent transfections
and are normalized to the wild type 3P enhancer reporter
gene.
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 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.
Figure 8:
A GAL4-Elk1 fusion protein responds to
multiple signal transduction pathways in GH 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.
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 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.
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.