Department of Pharmacology Case Western Reserve University School of Medicine Cleveland, Ohio 44106
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ABSTRACT |
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INTRODUCTION |
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Significant effort has been devoted toward understanding the regulatory
mechanisms controlling the biosynthesis of LH, a heterodimeric protein
composed of a common -subunit and unique ß-subunit that confers
biological specificity (1, 2). Many of the elements and factors that
are essential for expression of the common
-subunit gene have been
studied extensively in vitro (6, 7, 8, 9, 10, 11, 12) and in transgenic mice
(6, 13, 14, 15). From this work, it is clear that a complex array of
regulatory elements form a combinatorial code that directs expression
of the
-subunit gene to gonadotropes and conveys responsiveness to
GnRH. In contrast, while characterization of the LHß promoter has
lagged behind that of the
-subunit promoter, recent evidence
suggests that distinct arrays of regulatory elements confer gonadotrope
specificity and GnRH responsiveness to each gene.
All of the regulatory elements that have been identified in the
promoter of the LHß subunit gene lie within 500 bp of the start site
of transcription. This broad region can be further subdivided into two
distinct domains, proximal and distal (Fig. 1). Functional elements located in the
distal domain appear to differ across species. For example, CCAAT boxes
that bind NF-Y have been identified in the bovine LHß promoter
(16), whereas Sp1 binding elements occupy the distal domain of the rat
LHß promoter (17, 18, 19). These upstream elements of the distal domain
enhance promoter activity and, in the case of Sp1, contribute to GnRH
responsiveness (17, 18, 19, 20). The Sp1 elements do not, however, appear to be
essential for promoter activity or GnRH responsiveness, since
appreciable activity remains when they are deleted (17, 19).
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Closely associated with the GSEs is a pair of regulatory elements that bind early growth response protein 1 (Egr-1), also known as NGFI-A, zif/268, and Krox-24. These cis-acting elements have been studied extensively in the bovine (27), racine (26), and equine (25) LHß promoters and are necessary for full transcriptional activity as well as GnRH responsiveness (22, 25, 27). Through Egr-1, LHß mRNA levels are increased after hormonal activation of protein kinase C (PKC), a mediator of GnRH action (22, 27). Egr-1 is up-regulated and phosphorylated by the GnRH signaling cascade (31), suggesting that it is a downstream effector of the neurohormone. Further, SF-1 and Egr-1 functionally cooperate to increase LHß promoter activity, and mutation of any of the corresponding response elements attenuates this synergism (22, 27, 31). Targeted inactivation of the Egr-1 gene in mice resulted in a specific failure to synthesize LHß, although the number of gonadotropes and concentration of FSH were similar to those of wild-type animals (32, 33). Together, these data suggest that Egr-1, although not involved in differentiation of gonadotropes, is essential for expression of the LHß gene (33) and probably a direct target of the GnRH signaling pathway.
Nested between the pairs of Egr-1 and SF-1 binding elements in the
proximal domain of the LHß promoter is a single binding site for
Pitx1 (34). Pitx1, also known as Ptx1 (pituitary homeobox 1), p-OTX
(pituitary OTX-related factor), Bft (backfoot), and Brx2 (Rieg gene),
is a member of the bicoid-related subclass of homeobox genes
(35). The Pitx family of transcription factors includes three highly
conserved vertebrate paralogs that have been cloned in multiple
species: Pitx1, Pitx2, and Pitx3 (35). These transcription factors play
crucial roles in several aspects of development such as limb patterning
(Pitx1) (36, 37, 38), left-right axis determination (Pitx2) (39, 40, 41), and
proper eye formation (Pitx2 and Pitx3) (42, 43). In humans, genetic
mutations of Pitx1 are thought to be involved in development of
Treacher Collins Franceschetti Syndrome (44). Mutations of Pitx2 cause
Riegers syndrome (45), whereas Pitx3 mutations result in anterior
segment mesenchymal dysgenesis and cataracts (43, 45). Although both
Pitx1 and Pitx2 are early markers of pituitary development (35, 46),
Pitx1 also activates transcription of a large number of pituitary
target genes, including many that are active in gonadotropes, such as
those that encode , FSHß, LHß, and GnRH receptor (36, 47). Thus,
normal gonadotrope function clearly requires Pitx1.
Since Pitx1 activates transcription of the LHß gene, it is tempting to assume that this occurs through a DNA-dependent interaction. In this regard, three potential Pitx1 binding sites have been identified in the bovine LHß promoter (34). Of these, the best studied is the most promoter-proximal site that resides between the tandem Egr-1 and SF-1 sites. Overexpression of Pitx1 in heterologous cell lines activates the LHß promoter through a cooperative mechanism that includes direct interactions with SF-1 and Egr-1 (27, 28). Interestingly, however, this cooperative interaction appears to occur independently of Pitx1 binding to DNA (28). Instead, Drouin and colleagues (28) suggest that Pitx1 functions as an activator of SF-1. In addition to interacting with SF-1, Pitx1 may also augment GnRH responsiveness since it can interact with Egr-1, one of the downstream effectors of this pathway.
Given the strong conservation of the Pitx1 binding site across several species, we speculated that overexpression studies in heterologous cell lines might mask its importance in contributing to activity of the LHß promoter in more physiological settings such as homologous cell lines or transgenic mice where transcription factors are expressed at normal levels. We also wanted to determine whether the Pitx1 binding site contributes to GnRH responsiveness directly (i.e. in the absence of additional factors) or indirectly by supporting the action of Egr-1. Herein, we report results that address both questions using transfection and transgenic approaches that obviate the need for overexpression paradigms.
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RESULTS |
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Reporter activity was measured in LßT2 cells after transient
transfection of a mutant form of the LHß promoter in which the core
homeobox binding motif (ATTA) was converted to an RsaI
restriction enzyme site (GTAC). The LßT2 cells were chosen for these
analyses because they are one of the few cell lines that closely
resemble a differentiated gonadotrope (48). As illustrated in Fig. 2A, the wild-type LHß promoter, which
contains a functional Pitx1 binding site, displays activity 5-fold over
the promoterless control vector in LßT2 cells incubated without GnRH.
The Pitx1 cis-acting element contributes substantially to
this activity because luciferase activity in cells transfected with the
mutant Pitx1 LHß promoter was not different from that observed with
the promoterless control (P < 0.05). Given the
endogenous expression of SF-1 and Pitx1, these data suggest that the
Pitx1 site may be necessary for maintaining their appropriate
interaction (27).
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To confirm that mutating the core motif (ATTA) within the Pitx1 element
would inhibit the binding of Pitx1 or related proteins to DNA,
electrophoretic mobility shift analyses (EMSAs) were performed using a
double-stranded wild-type Pitx1 element as a radiolabeled probe and
nuclear extracts from LßT2 cells (Fig. 2B). Three bands representing
proteins that bind specifically to the Pitx1 element were observed
(bands 1, 2, and 4). Formation of these complexes was blocked by
addition of increasing molar concentrations (10x to 250x) of
unlabeled homologous competitor (wild-type Pitx1; lanes 25). While
the addition of increasing molar concentrations (10x to 250x) of the
unlabeled mutated Pitx1 competitor (lanes 69) also inhibited binding
to the labeled probe, the competition was not as efficient as the
wild-type Pitx1 site, indicating that mutation of the core
homeobox-binding motif substantially reduces its binding affinity.
Indeed, EMSA using radiolabeled probe representing the mutant form
of the Pitx1-regulatory element identified no protein
from LßT2 cell nuclear extracts that bound specifically and with high
affinity to this site (data not shown). To confirm the specificity of
these binding events, a nonradiolabeled double-stranded DNA
representing the distal NF-Y site that has been characterized in the
LHß promoter (16) was included (lane 10); this heterologous
competitor (250x) did not appear to affect any of the bound proteins.
An additional band that is nonspecific was also observed (band 3); the
intensity of this band did not change dramatically between any of the
samples. Based on these observations, we conclude that mutation of the
Pitx1 core sequence (ATTA) eliminates proteins that normally bind with
high affinity to this site.
It is unclear which shifted band represents Pitx1, as our attempts at detection with Pitx1 antibodies were unsuccessful for reasons that remain unexplained. However, because bacterially produced Pitx1 has been shown previously to bind the proximal Pitx1 site of the bovine LHß gene (28), we presume that at least one of the three bands represents Pitx1 and/or one of its isoforms. Indeed, several members of this family have been shown to have in vitro DNA binding specificities similar to that of Pitx1 (49) and can activate transcription of pituitary promoters, including that of the LHß gene (49). In fact, it is also possible that additional homeodomain proteins found in LßT2 cells can bind the Pitx1 site and affect transcription. While additional experiments will be necessary to define the identity of each protein complex detected by EMSA, the tight correlation between the absence of detectable binding with the mutant Pitx1 site and its loss of activity after transfection suggests that the binding of at least one of them is required for promoter activity.
The apparent lack of impact of the Pitx1 mutation on GnRH
responsiveness of the LHß promoter contrasts with other LHß
promoter mutations that have been assessed in either heterologous or
gonadotrope- derived cell lines (19, 25, 27). Individual mutations
in SF-1, Egr-1, or Sp1 binding sites often attenuate GnRH
responsiveness while pairwise mutations have an even greater impact
(17, 19, 25). In contrast, our data suggest that GnRH responsiveness of
the LHß promoter in LßT2 cells does not require a functional Pitx1
binding site and that Pitx1 may not be a direct target of the signaling
pathway. This is further supported by the unchanging expression of the
endogenous Pitx1 gene in T3-1 cells treated with forskolin, cyclic
ADP-ribose, and GnRH (27). Although this does not rule out the
possibility of post-transcriptional or translational effects of GnRH on
Pitx1 mRNA or protein, it does distinguish Pitx1 and its
cis-acting element from other downstream effectors that may
be direct targets of the GnRH signaling pathway.
The Pitx1-Regulatory Element of the LHß Promoter Fails to
Function as an Autonomous GnRH-Responsive Element in LßT2 Cells
In cell culture, the Pitx1 site may not be necessary for GnRH
responsiveness of the LHß gene. However, it has previously been shown
that Pitx1 functionally interacts with Egr-1 and SF-1, transcription
factors that bind the proximal promoter of the LHß gene and mediate
its response to GnRH. This bridging occurs even in the absence of the
consensus Pitx1 binding site in CV-1 cells (27). Thus, although Pitx1
may be necessary for induction of the hormonal response of GnRH on the
LHß gene, its cognate binding site appears to be dispensable. To
further clarify the role of the Pitx1 site as well as other regulatory
elements located in the proximal domain in mediating GnRH-induced
expression of the LHß gene, we determined which elements could
function autonomously as GnRH-responsive elements when attached to a
minimal heterologous promoter. Tandem copies of individual elements
that have been characterized in the proximal domain of the LHß
promoter were subcloned upstream of the PRL minimal promoter (50)
within a luciferase reporter vector. The gonadotrope-derived LßT2
cell line was transiently transfected with each construct and GnRH
responsiveness was measured (Fig. 3).
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Neither the proximal nor the distal SF-1 binding elements in the LHß
promoter appear to function as autonomous GnRH response elements (Fig. 3). In contrast, mutation of the distal SF-1 site renders the cognate
promoter nonresponsive to GnRH in transgenic mice (21), and mutation of
one or both SF-1 sites substantially decreases the GnRH-mediated
response in
T3-1 and LßT2 cells (25, 31). This suggests that while
SF-1 may not be a direct target of the GnRH signaling pathway, it must
play an indirect role in mediating the effects of the hormone through
its cooperative interaction with Egr-1 and Pitx1.
Tandem copies of the Pitx1 element linked to the PRL minimal promoter were also nonresponsive to GnRH treatment in transiently transfected LßT2 cells. Thus, like the elements that bind SF-1, the homeobox motif that binds Pitx1 may not function as an autonomous GnRH-responsive element. Based on the transfection studies described above, and coupled with the observation that Pitx1, whose expression levels do not appear to correlate with GnRH induction of gonadotropes (27), it is tempting to speculate that the Pitx1 site is not essential for GnRH-stimulated expression of the LHß gene. To test this notion further, we examined the activity of our LHß promoter constructs in transgenic mice where selected contributions of the hypothalamic-pituitary-gonadal axis could be assessed in a more physiological setting.
Mutating the Pitx1 Element within the LHß Promoter Abrogates
Pituitary Chloramphenicol Acetyltransferase (CAT) Activity in
Transgenic Mice
To assess the functional importance of the Pitx1 homeobox
core motif in the context of the full-length LHß promoter within a
physiological context, the same mutant and wild-type promoter
constructs used in vitro were linked to a CAT reporter and
used to develop several lines of transgenic mice. We have shown
previously that the full-length wild-type LHß promoter targets
expression of reporter genes specifically to gonadotropes in transgenic
mice and renders them responsive to GnRH and gonadal steroids (16, 21, 51). In contrast to the robust activity observed with the wild-type
promoter (wt-2), pituitary CAT activity in the Pitx1 mutant lines of
transgenic mice was indistinguishable from activity in the pituitaries
of nontransgenic mice (Fig. 4). This
pattern of expression, or lack thereof, was seen in both males and
females (P < 0.01).
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Increasing Endogenous GnRH by Ovariectomy Does Not Rescue Pituitary
CAT Activity in Transgenic Mice Harboring the Mutated Pitx1 Element
within the LHß Promoter
Expression of the LHß gene responds robustly to GnRH and the
homeobox binding motif appears to be unnecessary for this response in
cultured cells. Thus, we reasoned that increasing endogenous GnRH
within physiological limits might increase pituitary transgene activity
to detectable levels in animals harboring the mutated form of the LHß
construct. Because GnRH is secreted in regular pulses, administration
of exogenous pulsatile GnRH is difficult. Therefore, we elected to
increase GnRH levels through ovariectomy of randomly cycling transgenic
females (Fig. 5). Removal of the ovaries
eliminates steroid negative feedback, allowing a sustained increase in
synthesis and secretion of GnRH in a pulsatile manner from the
hypothalamus (2). Intact animals were not included in these experiments
because their asynchronous reproductive cycles result in wide
variations in serum LH concentrations that would require large numbers
of animals to assure statistical differences at each stage of the
estrous cycle. Instead, we treated a subset of gonadectomized
females with a specific GnRH antagonist, antide, to provide an indirect
measure of GnRH responsiveness. As shown in Fig. 5A
, serum LH
concentrations were considerably higher in all ovariectomized
transgenic mice when compared with their antide-treated ovariectomized
counterparts. This provides evidence for the effectiveness of antide in
blocking events normally mediated by GnRH.
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Antide also lowered activity of the wild-type transgene in
ovariectomized females when compared with their vehicle-treated
counterparts (Fig. 5B; P < 0.01). This provides an
index of the GnRH responsiveness conferred by the transgenic wild-type
LHß promoter. In contrast, the Pitx1 mutant transgene remained
inactive in both lines of ovariectomized females (Fig. 5B
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suggests that the mutated LHß promoter could not be rescued by high
physiological concentrations of GnRH in transgenic mice. In fact,
levels of expression of the mutant transgene remained undetectable as
demonstrated by comparison to activity of the wild-type transgene in
nonexpressing tissues. Thus, in the absence of the 4-bp core homeobox
motif, activity of the LHß promoter is minimal and cannot be rescued
by physiologically elevated concentrations of GnRH.
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DISCUSSION |
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We have reassessed the importance of this Pitx1 site using two experimental models in which expression of the endogenous LHß gene occurs in concert with the three DNA-binding proteins of interest: Egr-1, Pitx1, and SF-1. Collectively, our data indicate that the proximal Pitx1 site is essential for LHß promoter activity. Mechanistically, this suggests that interaction with DNA causes an allosteric change that endows endogenous Pitx1 with the ability to carry out a transcriptional program specifically adapted for the LHß promoter. This also indicates that while the other putative Pitx1 binding sites in the LHß gene may be functional, they are clearly not sufficient to support Pitx1-mediated activation of the promoter. Even physiologically high concentrations of endogenous GnRH reached by removal of gonadal steroid negative feedback could not activate the Pitx1 mutant form of the promoter in transgenic mice.
Because expression of the endogenous LHß gene is exquisitely dependent upon GnRH, the role of the Pitx1 binding site in mediating responsiveness to this neurohormone requires further consideration. While our experimental approaches support the importance of the Pitx1 site in defining a fully functional LHß promoter, we also uncovered an unexpected dichotomy between the necessity for the Pitx1 element in conferring a GnRH response of the LHß promoter in cell lines vs. transgenic mice. There are many possible explanations for this contrariety including 1) differences in pulse patterns of GnRH and its concentration (52, 53); 2) differences in thresholds of detection associated with transfection and transgenics; 3) a generalized variation in the expression of transcription factors between the in vitro and in vivo settings; and 4) simple differences in mass action. Based on these possibilities, we suggest that the transfection assays reveal the potential for GnRH to rescue at least some activity of the mutant LHß promoter, possibly through the action of Egr-1, but that this rescued activity remains below the limit of detection in transgenic mice.
While the use of overexpression paradigms in heterologous cells appear
to have masked the importance of the Pitx1 site, they have been
extremely valuable in demonstrating potential interactions that occur
between Pitx1, Egr-1, and SF-1, contributions that cannot be
ascertained from the experimental approaches we used. For example,
Tremblay et al. (28) showed direct cooperative interactions
between Pitx1 and SF-1 and proposed that Pitx1 may serve as an
efficient ligand for SF-1 by modulating its activity in gonadotropes. A
constitutively-active form of SF-1 in which the ligand-binding domain
has been deleted (SF-1LBD) activated the LHß promoter more than
the wild-type SF-1, and, unlike wild-type SF-1, SF-1
LBD was unable
to synergize with Pitx1, although the transcription factors were still
shown to interact. In fact, the activities of SF-1 with Pitx1 compared
with SF-1
LBD alone were identical. Collectively, the extent of
the interaction between the transcription factors offers broad support
for the possibility that Pitx1 serves as a physiological ligand that
activates SF-1. If Pitx1 is a functional ligand for SF-1, then our data
suggest that this property may only occur when Pitx1 is bound to its
element.
Like the Pitx1 element, the SF-1 element does not act as an autonomous GnRH response element when linked in tandem to a heterologous promoter. In fact, neither the proximal nor the distal SF-1 binding elements in the LHß promoter appear to confer autonomous GnRH responsiveness. In contrast, our previous studies in transgenic mice indicated that the distal SF-1 binding site, like the proximal Pitx1 site, may be essential for GnRH-regulated expression of LHß (21) because activity of a transgene harboring a mutated distal SF-1 site remained refractory to a postcastration rise in GnRH. Taken together, these apparently contradicting data may indicate that while the SF-1 elements do not act as autonomous GnRH response elements, they may be regarded as critical accessory elements indirectly involved in the response. Indeed, it has been shown that while SF-1 does not have a direct effect on GnRH responsiveness of the LHß promoter, the ability of Egr-1 to activate the LHß promoter in response to GnRH requires an direct interaction between Egr-1 and DNA-bound SF-1 (19, 26, 27, 31). In this regard, the SF-1 sites, like the Pitx1 element, may be critical for a normal GnRH response even though they do not serve as direct downstream targets of the signaling pathway.
In contrast to the Pitx1 and SF-1 elements, the Egr-1 binding sites act as autonomous GnRH response elements when placed in tandem on a heterologous promoter. Binding of GnRH to receptors at the cell surface of gonadotropes activates the PKC signaling pathway (54, 55), which leads to elevated Egr-1 mRNA levels (22, 31) as well as phosphorylation of Egr-1 protein (27). In the absence of GnRH, only low levels of monophosphorylated Egr-1 can be detected (27). Egr-1 is vital for expression of LHß, as mice lacking this transcription factor are devoid of LH, but have normal serum FSH concentrations and gonadotrope numbers (32, 33). SF-1 and Pitx1 are also essential for GnRH responsiveness, because enhancement of Egr-1 action by superphysiological levels of GnRH (seen postcastration) is insufficient for expression of LHß in the face of a mutant binding site for either SF-1 (21) or Pitx1. Several groups have shown the importance of synergy between Egr-1 and SF-1 for activation of the LHß gene by GnRH (19, 22, 25, 27, 31). In addition, Tremblay and Drouin (27) have firmly established the role of Pitx1 in enhancing this cooperative interaction. However, while the site at which Pitx1 binds does not appear to be necessary for this synergism when transcription factors are overexpressed in heterologous cell lines, the site is crucial for expression of the LHß promoter in transgenic mice, as observed by the absence of detectable transgene expression in mice presented herein, even in the presence of high endogenous GnRH, and presumably increased Egr-1.
In contrast to the regulatory elements in the proximal domain, upstream DNA cis-acting elements in the distal domain display species-specific diversity, suggesting that multiple configurations are capable of achieving the same end point. For example, the distal domain of the rat LHß gene harbors two adjacent Sp1 binding sites (17). The ubiquitous Sp1 transcription factor binds these sites and enhances GnRH-stimulated expression (17) through interactions with factors that bind elements in the proximal promoter, including SF-1 and Egr-1 (18, 19). Indeed, Kaiser et al. (19) have described these elements as forming a "tripartite GnRH response element." In considering the proposed role for Sp1, it is important to note that the Sp1 elements are less conserved than the proximal LHß promoter elements, suggesting that their importance might be limited to the rat LHß promoter. For example, the bovine LHß promoter lacks Sp1 sites at positions comparable to those found in the rat LHß promoter. Rather, a CCAAT box that binds NF-Y is located in the distal domain of the bovine LHß promoter that contributes to its activity. This site, however, does not appear to be required for GnRH responsiveness (16). Therefore, from studies performed to date, the role of the distal domain in mediating responsiveness to GnRH appears species specific and requires further investigation.
In summary, we suggest that the strong conservation of regulatory
elements in the proximal region of the LHß promoter across several
species underscores their importance in regulating transcription, and,
in particular, responsiveness to GnRH. In Fig. 6 we diagram a model
that builds on the work from several laboratories (22, 26, 27, 28, 31) as
well as the work reported herein. We propose that the promoter-proximal
elements define a core "composite GnRH response element" that
includes the essential Pitx1 binding site as well as four other highly
conserved regulatory elements (pEgr-1, pSF-1, dEgr-1, and dSF-1).
Transcription factors that bind to this region interact directly with
one another and most likely with transcription factors that bind to
elements in the distal region such as Sp1 for the rat promoter (17, 19)
and NF-Y for the bovine promoter (16). While Pitx1 has yet to be tested
for functional synergism with proteins that bind to the distal
elements, it can cooperatively interact with both Egr-1 and SF-1 (27)
and may serve as a physiological ligand for the latter (28). More
importantly, activity of the LHß promoter requires the homeobox
binding motif that defines the core of the Pitx1 cis-acting
element. Binding of Pitx1, and perhaps other related homeobox proteins,
to this element increases its effectiveness in acting as a functional
partner for SF-1 and Egr-1. Although Egr-1 can be regarded as a direct
downstream effector of the GnRH signal pathway, DNA-bound Pitx1 and
SF-1 are required accessory factors as elimination of their binding
sites renders the promoter inactive and, in vivo,
unresponsive to increased physiological levels of the neurohormone.
Thus, all of the aforementioned elements serve as vital docking sites
required for the formation of a higher-order transcriptional complex
that directs spatial expression and hormone responsiveness of the LHß
gene.
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MATERIALS AND METHODS |
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Multimerized regulatory elements in the forward direction linked to PRLpGL3 were generated by ligating double-stranded oligos containing the various regulatory sequences flanked by HindIII sites into the same site in the multiple cloning region of PRLpGL3: 2xpEgr1PRLpGL3 (5'-AGC TTT GCC GCC CCC ACA GCA-3'; 5'- AGC TTG CTG TGG GGG CGG CAA-3'), 2xpSF1PRLpGL3 (5'-AGC TTC GGC GGC CTT GCC GCA-3'; 5'-AGC TTG CGG CAA GGC CGC CGA-3'), 2xPitx1PRLpGL3 (AGC TTG GGA GAT TAG TGA AGC TTG GGA GAT TAG TGA-3'; 5'-AGC TTC ACT AAT CTC CCA AGC TTC ACT AAT CTC CCA-3'), 2xdEgr1PRLpGL3 (5'-AGC TTT CTC GCC CCC GGG GAG A-3'; 5'-AGC TTC TCC CCG GGG GCG AGA A-3'), and 2xdSF1PRLpGL3 (5'-AGC TTC CCT GAC CTT GTC TA-3'; 5'-AGC TTA GAC AAG GTC AGG GA-3').
The Pitx1 mutant form of an LHß promoter-driven CAT reporter (µPitx1-LHßCAT) vector that was used to generate transgenic mice in these experiments was constructed by subcloning the bovine LHß promoter insert that contained the mutated Pitx1 site (as described above) into the HindIII site of the BSK-(-776/+10)bLHßCAT vector, as has been described previously (51), replacing the wild-type promoter with the mutated form. All clones were confirmed with dideoxynucleotide sequencing.
EMSAs
LßT2 cells were treated with GnRH (100 nM) for
24 h before extraction of nuclear proteins. Nuclear extracts were
prepared as described previously (8). EMSAs were performed essentially
as described (21) except with 23 µg of LßT2 nuclear protein. The
following oligonucleotides were used: Pitx1(+), 5'-CCG GGG AGA
TTA GTG TCC AGG TTA CCC CAC-3' (core homeobox motif
underlined); Pitx1(-), 5'-GTG GGG TAA CCT GGA CAC TAA
TCT CCC CGG 3' (core homeobox motif underlined); µPitx1(+),
5'-CCG GGG AGG TAC GTG TCC AGG TTA CCC CAC- 3' (mutated
motif underlined); µPitx1(-), 5'-GTG GGG TAA CCT GGA CAC
GTA CCT CCC CGG-3' (mutated motif underlined);
dNF-Y(+), 5'-CTG CAG CCA ATC ACC ATC GGA AAA TGG AGC T-3'; dNF-Y(-),
5'-CCA TTT TCC GAT GGT GAT TGG CTG CAG AGC T-3' (16). Double-stranded
oligodeoxynucleotides were end labeled with
[-32P] ATP (NEN Life Science Products, Boston, MA) using T4 polynucleotide kinase (Life Technologies, Inc., Gaithersburg, MD). Poly(dI-dC) was purchased
from Roche Molecular Biochemicals (Indianapolis, IN).
Cell Culture and Transient Expression Assays
Transient transfection studies were performed in LßT2 cells,
which were maintained in high-glucose DMEM containing 2 mM
L-glutamine and supplemented with 10% FBS and antibiotics.
The day before transfection, cells were plated at a density of
approximately 2 x 106 cells per 35-mm well.
Transfections were carried out using media lacking serum and
antibiotics with 10 µl LipofectAMINE reagent (Life Technologies, Inc.), 2.0 µg each test vector, and 100 ng
pRL-CMV (Promega Corp., Madison, WI), which was used to
normalize data for transfection efficiency. Cell cultures were
incubated with the transfection mixtures for approximately 18 h at
37 C in a humidified atmosphere with 5% CO2.
After incubation, complete media were added to the cells, which, where
indicated, were also supplemented with 100 nM GnRH.
Twenty-four hours after the addition of fresh media and hormonal
treatments, cells were lysed in passive lysis buffer (Promega Corp.), and a dual-luciferase assay was performed on each
cellular lysate as per standard procedures. Transient transfections
were performed a minimum of three times with at least two separate
plasmid preparations for each construct that was tested. Luciferase
activity was analyzed by two-way ANOVA (Fig. 2) or one-way ANOVA (Fig. 3
), and differences among treatments were determined by the post-hoc
test, Tukeys Honestly Significant Difference, a very conservative
pairwise comparison test.
Transgenic Mice
The µPitx1-LHßCAT insert was liberated from the vector using
a SalI/BamHI digest. Insert DNA was purified by
0.7% agarose gel electrophoresis. Transgenic mice were generated as
previously described (7). Mice were genotyped by PCR to amplify DNA
found within the transgene. Primers that amplified a fragment of the
endogenous murine ß-globin gene were also included in the PCR to
confirm the integrity of genomic DNA within each reaction. Mice were
bred to obtain single integration sites of the transgene, as determined
by Mendelian inheritance patterns. All mice were housed in
microisolator-plus units under pathogen-free conditions. Food and water
were provided ad libitum, and animals were subjected to
a 12-h light, 12-h dark cycle. Mice harboring the wt-LHßCAT
transgene have been previously reported (16, 21).
Adult pituitaries were immersed in 200 µl 0.25 M Tris (pH 7.8). Pituitary lysates were obtained and CAT assays were performed as described previously (16, 21, 51). In all assays, 25 µg pituitary protein were used, and the assays were incubated for 2 h (wt-LHßCAT transgenic mice) or 18 h (µPitx1-LHßCAT and nontransgenic mice) and plotted as percent conversion/µg protein/h. The increased incubation for pituitary tissues extracted from both nontransgenic and µPitx1-LHßCAT mice has been shown to increase assay sensitivity in previous experiments (16), allowing for determination of low vs. no CAT activity. Radiolabeled chloramphenicol was obtained from NEN Life Science Products, and acetyl coenzyme A was purchased from Sigma (St. Louis, MO).
To examine GnRH responsiveness, sexually mature mice were ovariectomized under avertin anesthesia. Immediately after surgery, 300 µl antide (200 ng/ml; from Sigma) or vehicle (20% propylene glycol in normal saline) was injected subcutaneously (21). Additional injections were given every 48 h for a total of 10 days. On the tenth day, animals were killed and tissues and blood were collected. Serum LH concentrations were measured using a previously validated RIA (28). CAT activity was analyzed by two-way ANOVA. Differences among treatment groups were determined by Tukeys Honestly Significant Difference.
Experimental Animals
All animal studies were conducted in accord with the principles
and procedures approved by the Institutional Animal Care and Use
Committee of Case Western Reserve University.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant DK-28559 (to J.H.N.) and NIH National Research Service Award Fellowship DK-09843 (to C.C.Q.).
Received for publication August 7, 2000. Revision received December 12, 2000. Accepted for publication December 20, 2000.
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