| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Animal Reproduction and Biotechnology Laboratory (D.L.D., T.M.N., C.M.C.), Department of Physiology, Colorado State University, Fort Collins, Colorado 80523; Department of Pharmacology (C.C.Q.), Case Western Reserve University School of Medicine, Cleveland, Ohio 44106; USDA-CSREES-NRI (D.L.H.), Washington, DC 200250–2241
Address all correspondence and requests for reprints to: Colin M. Clay, ARBL-Foothills Campus, Colorado State University, Fort Collins, Colorado 80523. E-mail: cclay{at}cvmbs.colostate.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To address the molecular mechanisms underlying cell-specific and
hormonal regulation of GnRHR gene expression, we have cloned both the
murine and ovine GnRHR genes (16, 17). We have found that 1,900 bp of
5' flanking sequence from the murine GnRHR gene is both responsive to
GnRH stimulation and sufficient to direct expression of luciferase to
the pituitary, brain, and testes of transgenic mice (18). More
recently, 1,200 bp of 5'flanking sequence from the murine GnRHR gene
was used to target expression of SV40 large T antigen to gonadotrope
cells in transgenic mice (19). Based on in vitro studies
using the gonadotrope-derived 
T3–1 cell line, we have suggested
that cell-specific expression is dependent on a tripartite enhancer
that consists of a binding site for steroidogenic factor-1 (SF-1), an
AP-1 element, and an element we have termed the GnRH
receptor activating sequence (GRAS)
(16, 20, 21). In addition to contributing to "basal" promoter
activity, this tripartite enhancer also mediates multiple endocrine
inputs. Specifically, GnRH responsiveness of the GnRHR gene is
partially mediated by protein kinase C activation at AP-1 (22, 23),
whereas activin/follistatin responsiveness is mediated at GRAS
(24).
In contrast to GnRH and activin regulation of the murine GnRHR gene, virtually nothing is known about the mechanisms underlying estradiol regulation of GnRH receptor expression. This is particularly troubling because estradiol is generally conceded to be perhaps the primary regulator of GnRH receptor numbers on gonadotrope cells. This phenomenon has received particular attention as it is thought to be a central event underlying increased pituitary sensitivity to GnRH during the preovulatory period and thus contributes to the high rates of LH secretion necessary for ovulation (6, 9, 25, 26, 27, 28). Subsequent to the availability of complementary DNAs (cDNAs) encoding the GnRH receptor, a number of studies have demonstrated coordinate changes in GnRH receptor messenger RNA (mRNA) and GnRH receptor numbers associated with estradiol treatment (12, 14, 29, 30, 31, 32, 33). While it is possible that estradiol exerts its stimulatory effects on GnRHR expression via a posttranscriptional mechanism such as mRNA stabilization, the ability of actinomycin D to block estradiol up-regulation of GnRH receptor numbers in primary cultures of ovine pituitary cells does not support this contention (29). Alternatively, estradiol might directly stimulate GnRHR gene expression. Unfortunately, a canonical estradiol response element (ERE) has not been identified in any GnRHR gene reported to date (16, 17, 34, 35, 36), nor have we been able to detect estradiol regulation of the murine GnRHR gene in either transient transfection paradigms or transgenic mice (18). Thus, despite the central role of estradiol in regulation of the GnRHR gene, the underlying mechanisms remain undefined.
Herein, we report the construction of transgenic mice harboring a chimeric ovine GnRHR promoter-luciferase gene that is robustly responsive to estradiol. This represents the first evidence of direct estradiol activation of a gene promoter in gonadotropes of the anterior pituitary gland. Furthermore, it is striking to note that the promoter of the ovine GnRHR gene is unresponsive to estradiol in both gonadotrope and nongonadotrope-derived cell lines, suggesting that transgenic mice may represent the only viable avenue for investigating molecular mechanisms underlying estradiol regulation of GnRHR gene expression.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vector construction
Plasmid oGR-2700LUC was constructed by PCR amplification of the
5'-flanking region contained in an ovine GnRH receptor genomic clone
using gene-specific primers (sense: 5'-GGCACTTGCCATCTA-3'; antisense:
5'-GAGCTCGGCACTTCTGATGTT-3'). Following amplification, 1U
Taq polymerase was added to the reaction and incubated at 72
C for 10 min, and the resulting product was ligated into pCRII using TA
cloning methodologies (Invitrogen). The 2,700-bp PCR
product was released from pCRII by digestion with KpnI and
XhoI and ligated into the same sites of the pGL3 luciferase
expression vector (Promega Corp., Madison, WI).
Plasmid oGR-9100LUC was constructed by adding additional 5' flanking sequence to poGR-2700LUC. Briefly, poGR-2700LUC was completely digested with KpnI and partially digested with XbaI to remove the 5' terminal 1,100 bp of the promoter. Additional 5'flanking sequence was prepared by digesting an ovine genomic clone with KpnI and XbaI to excise 7,500 bp of sequence 5' to the promoter sequenced retained in the digested poGR-2700 vector. The insert was then ligated into the prepared poGR-2700LUC vector.
Plasmid oGR-160LUC was constructed by PCR amplification of the 5'flanking region contained in an oGnRHR genomic clone using the antisense primer used for the construction of poGR-2700LUC and a gene-specific primer located 160 bp 5' of the start site of translation (5'- TTAAATACAAAGTATCTCAGG-3').
Cell culture and transient transfections
All cell cultures were maintained in a humidified atmosphere of
5% CO2 at 37 C. T3–1 cells were cultured in
high-glucose DMEM containing 2 mM glutamine, 5% FBS, 5%
horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate.
COS-7 and LßT2 cells were cultured in high-glucose DMEM containing 2
mM glutamine, 10% FBS, 100 U/ml penicillin, and 100
µg/ml streptomycin sulfate. MCF-7 cells were maintained in
high-glucose DMEM containing 2 mM glutamine, 10% FBS, 1
mM sodium pyruvate, 10 µg/ml insulin, 100 U/ml
penicillin, and 100 µg/ml streptomycin sulfate. To examine basal
activity and estradiol responsiveness, the plasmids were transfected
using the lipofectamine procedure (Life Technologies, Inc., Gaithersburg, MD) as previously described (21). On the day
before transfection, 0.5–3 x 106 cells
were seeded into a 35-mm wells of a 6-well tissue culture plate.
Plasmid DNA was combined with lipofectamine reagent in 200 µl of
high-glucose DMEM containing 2 mM glutamine (transfection
media) and incubated at RT for 30 min. The mixtures were diluted to 1
ml with the transfection media and applied to cell cultures previously
washed with transfection media. Where indicated, cultures were treated.
After incubation for approximately 16 h in a humidified atmosphere
of 5% CO2 at 37 C, the cell cultures were
supplemented with 1 ml of high-glucose DMEM containing 2 mM
glutamine, 10% FBS, 10% horse serum, 200 U/ml penicillin, and 200
µg/ml streptomycin sulfate and, where indicated, additional
treatments were added. Approximately 24 h after transfection, the
media was aspirated and the cells were washed twice with ice-cold PBS
(pH 7.4). Cells were lysed in the wells by the addition of 200 µl
lysis buffer (0.025 M glycyl-glycine, pH 7.8; 1
mM dithiothreitol, 15 mM
MgSO4, and 1.0% Triton X-100). Cellular debris
was removed by centrifugation at 16,000 x g for 2 min.
Cellular lysates were immediately assayed for luciferase activity by
adding 20 µl lysate to 100 µl luciferin substrate (Promega Corp.) and measuring luminescence with a Turner model TD-20E
luminometer set for a 5-sec delay and 10-sec integration.
ß-galactosidase activity was measured in 50 µl of lysate using the
luminescent assay system and substrate (Tropix, Bedford, MA) with the
same luminometer set for a 10-sec delay and 5-sec integration following
manufacturer’s instructions. Luciferase activity was normalized for
transfection efficiency by dividing the luciferase activity by
ß-galactosidase activity.
Generation and screening of transgenic mice
Fusion genes consisting of either approximately 2,700 or 9,100
bp of the 5'flanking region from the oGnRHR gene subcloned upstream of
the cDNA encoding firefly luciferase were released from poGR-2700LUC or
poGR-9100LUC by digestion with SacI and BamHI and
purified by electroelution. Transgenic founder animals were generated
by DNX, Inc. using standard microinjection techniques. Genomic DNA was
extracted from tail biopsies and analyzed for the presence of the
transgene by slotblot analysis or PCR amplification using primers
specific to luciferase. All mice used in these experiments were of the
FVB-inbred strain (38). Animals were maintained under a 14-h light,
10-h dark cycle and received food and water ad libitum. All
experiments were performed under veterinary supervision with approval
from the Colorado State University Animal Care and Use Committee and in
accordance with the NIH Animal Care and Use Guidelines. All experiments
were conducted using animals greater than 6 weeks of age.
Luciferase assays
Tissue extracts were prepared by homogenization in 200 µl cold
lysis buffer (25 mM glycyl-glycine, pH 7.8; 1.0% Triton
X-100, 10 mM MgSO4, and 1.0
mM dithiothreitol. Cellular debris was pelleted by
microcentrifugation at 16,000 x g for 5 min at 4 C.
Cellular lysates were immediately assayed for luciferase activity by
adding 20 µl lysate to 100 µl luciferin substrate (Promega Corp.) and measuring luminescence with a Turner model TD-20E
luminometer set for a 5-sec delay and 10-sec integration. Total protein
was precipitated from lysates with 10% trichloroacetic acid and then
dissolved in 0.1 N NaOH. Protein concentrations
were determined using bicinchoninic acid (BCA Assay, Pierce Chemical Co., Rockford, IL). Luciferase activity was adjusted
for protein content by dividing the arbitrary light units by the
protein content in mg.
Animal treatments
Exp 1: GnRH and estradiol regulation of oGR-9100LUC transgene
expression. Female transgenic mice were ovariectomized (OVX) and
separated into three groups. At ovariectomy, one group received sc
implants of slow-release 2.5 mg estradiol-17ß pellets (39), a second
group was left untreated to serve as the OVX control. After 4 days, two
groups, excluding the OVX control group, received a single ip injection
of 300 µl GnRH antiserum (AS). At 1 week following ovariectomy, the
groups (OVX, OVX + AS, OVX + AS + estradiol), were killed, and
pituitary, brain, and liver were harvested and assayed for luciferase
activity. Trunk blood was collected for analysis of serum
concentrations of LH (40). This study was repeated in castrated (CAS)
male transgenic mice. In the males, an additional group treated with AS
was also administered 1 ng of a noncross-reacting GnRH analog
(D-Ala-6-GnRH) ip in 100 µl 0.15 M NaCl every 2 h
for 48 h beginning immediately following injection of AS (40). Two
days later each group of animals (CAS, CAS + AS, CAS + AS + estradiol,
and CAS + AS + D-ala-6-GnRH) were killed and pituitary, brain, and
liver were harvested and assayed for luciferase activity. Trunk blood
samples were collected for analysis of serum concentrations of LH (40)
and estradiol concentrations (41).
Exp 2: Synergistic induction of pituitary luciferase by estradiol and GnRH in oGR-9100LUC transgenic mice. Female transgenic mice were ovariectomized and separated into three groups (OVX + AS + estradiol, OVX + AS + D-Ala-6-GnRH, OVX + AS + estradiol + D-Ala-6-GnRH). At ovariectomy, estradiol groups received sc implants of slow-release 2.5 mg estradiol-17ß pellets. After 4 days, each group received a single 300 µl ip injection of AS. Immediately following injection with AS, groups receiving D-Ala-6-GnRH were injected ip with 1 ng of D-Ala-6-GnRH in 100 µl of 0.15 M NaCl every 2 h for 48 h. At the end of the 48-h period and 1 week following ovariectomy, the animals were killed, and pituitary, brain, and liver were harvested and assayed for luciferase activity as above.
Exp 3: GnRH and estradiol regulation of oGR-2700LUC transgene expression. Female transgenic mice were ovariectomized and separated into three groups (OVX, OVX + AS, OVX + AS + estradiol). At OVX, one group received sc implants of slow-release 2.5 mg estradiol-17ß pellets. After 4 days, AS groups received a single 300-µl ip injection of AS. At 1 week following OVX, the animals were killed, and pituitary, brain, and liver were harvested and assayed for luciferase activity. Trunk blood was collected for analysis of serum concentrations of LH (40). Male transgenic mice were castrated and separated into three groups (CAS, CAS + AS, CAS + AS + D-Ala-6-GnRH). After 4 days, AS groups received a single 300-µl ip injection of AS. Immediately following injection with AS, the group receiving D-Ala-6-GnRH were injected with 1 ng of D-Ala-6-GnRH in 100 µl 0.15 M NaCl every 2 h for 48 h. At the end of the 48-h period and one week following CAS, the animals were killed, and pituitary, brain, and liver were harvested and assayed for luciferase activity. Trunk blood was collected for analysis of serum concentrations of LH (40).
Data analysis
Tissues expressing luciferase activity at levels above the mean
+ 2 SD of values in tissues from nontransgenic animals was
considered positive for expression of the transgene. Differences in
luciferase activity and serum concentrations of LH were determined by
one-way ANOVA using the general linear model procedure of SAS (42).
Where necessary, data were log transformed to correct for heterogeneity
of variance. Means were separated using Student’s t test
(see Figs. 2
and 5) and Dunnett’s (see Fig. 1) or Tukey’s (see Figs. 4, 6, and 8) methods of multiple comparisons.
|
|
|
|
|
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T3–1cell line. A luciferase reporter construct
containing two copies of the Xenopus vitellogenin estrogen response
element (43)(ERE, 5' AGGTCACAGTGACCT 3') inserted upstream of the
thymidine kinase minimal promoter (ERE-TKLUC) served as a positive
control for estradiol responsiveness. In stark contrast to the murine
promoter, which displays robust transcriptional activity in T3–1
cells (16, 20, 21), none of the oGnRHR promoter constructs displayed
transcriptional activity different from the promoterless control vector
(Fig. 1
). Similarly, no effect of
estradiol on transcriptional activity was evident for any of the ovine
promoter constructs tested. In contrast, the inclusion of the tandem
ERE dramatically increased (P < 0.05) activity of the
minimal TK promoter (TKLUC vs. ERE-TKLUC). Surprisingly,
estradiol treatment had no further stimulatory effect on activity of
ERE-TKLUC, suggesting that activation of transcription from an ERE in
T3–1 cells can occur in a ligand-independent fashion. Finally,
cotransfection of the LUC reporter constructs with HEO—an expression
vector for human estrogen receptor (ER) (44, 45), did not reveal any
additional increase in activity of ERE-TKLUC or any of the GnRHR
promoter constructs either in the presence or absence of estradiol
(data not shown). Thus, despite expression of the endogenous murine
GnRHR gene, neither basal activity nor estradiol responsiveness of the
oGnRHR promoter is revealed in T3–1 cells.
To further explore this issue, we expanded our analyses to another cell
line of gonadotrope origin (LßT2) and two nonpituitary cell lines
that either do (MCF-7) or do not (Cos-7) express ER. While estradiol
treatment led to an approximately 4-fold increase in activity of
ERE-TKLUC in LßT2 cells cotransfected with HEO, there was no effect
of estradiol on activity of any of the oGnRHR constructs tested (Fig. 2A). Similarly, estradiol responsiveness
of ERE-TKLUC was readily apparent in both MCF-7 and Cos-7 cells in
which ER was introduced by cotransfection with HEO; however, an
estradiol response of the oGnRHR promoter was not detected in either
cell line (Fig. 2, B and C).
Approximately 9,100 bp of 5' flanking region from the ovine GnRHR
gene directs tissue-specific expression in transgenic mice
The absence of basal activity of the oGnRHR promoter in the
gonadotrope-derived cell lines as well as the lack of estradiol
responsiveness suggests either the absence of critical cis-acting
elements located within 9,100 bp of 5' flanking region or the absence
of critical transcription factors in the cell lines examined. To
discriminate between these possibilities, we chose to determine if
activity of the oGnRHR promoter might be revealed in transgenic mice.
Toward this end, transgenic founder animals harboring a fusion gene
consisting of approximately 9,100 bp of 5'-flanking sequence from the
oGnRHR gene linked to the coding sequence for luciferase were
generated. F1 progeny representing three independent lines were used to
assess expression of the transgene (Fig. 3). Based on these studies, at least
three patterns emerged. First, luciferase expression was consistently
detected in the pituitary, brain, and gonads across all lines. Each of
these tissues are established sites of expression of the endogenous
GnRHR gene (46, 47, 48). Second, pituitary expression of luciferase was
higher in females than males. Finally, testicular expression of
luciferase was consistently higher than ovarian expression. While
luciferase expression was detected in other tissues, these patterns
were not consistent across all lines. Thus, despite the absence of
transcriptional activity in vitro, approximately 9,100 bp of
proximal 5' flanking region from the oGnRHR gene is sufficient to
direct tissue-specific expression of a heterologous reporter gene in
transgenic mice.
|
Immunoneutralization of GnRH reduced pituitary expression of luciferase
by approximately 80% and led to the expected decrease in serum
concentrations of LH (Fig. 4A). The
addition of estradiol increased pituitary and brain luciferase
expression by approximately 50-fold and 2-fold, respectively, over
animals receiving AS alone, but did not alter serum concentrations of
LH. Liver expression of luciferase was below background (mean + 2
SD of nontransgenic tissue) and was not affected by either
AS or estradiol treatment. Finally, estradiol replacement increased
serum estradiol levels to 749 ± 114 from 10.4 ± 3.2 pg/ml
and 12.6 ± 10.8 pg/ml (mean ± SD) for untreated
OVX and OVX + AS controls and increased uterine weights by
approximately 4-fold.
To determine if estradiol regulation of the 9,100 bp ovine GnRHR
promoter was unique to females, the same experimental paradigm was
employed using castrated, male transgenic mice with the exception of an
additional group that received both AS and 1 ng of a noncross-reacting
GnRH analog, D-Ala-6-GnRH (18), every 2 h for 48 h beginning
immediately following injection of AS. As with female mice, estradiol
treatment increased expression of the transgene in the pituitary and
brain (Fig. 4B). Similarly, antiserum reduced pituitary expression of
luciferase and serum concentrations of LH. Replacement with
D-Ala-6-GnRH in animals treated with antiserum restored both pituitary
luciferase expression and serum concentrations of LH to levels not
different from the castrate controls.
To confirm that estradiol regulation was not unique to a single line of
mice, we assessed estradiol responsiveness of the oGR-9100LUC fusion
gene in a second line established from founder no. 887. In this
experiment, we simply compared the effects of estradiol in the face of
GnRH immunoneutralization. As in the 884 line, estradiol treatment
resulted in an approximately 22-fold and 4-fold increase in luciferase
expression in the pituitary and brain, respectively (Fig. 5). One notable difference between the
two lines of transgenic mice was higher brain expression of luciferase
in the no. 887 line. This was most apparent following antiserum and
estradiol treatment in which the level of expression in the no. 887
animals was approximately 5-fold greater than that in the no. 884 line.
However, this higher level of expression in both estradiol treated and
untreated animals is consistent with the higher luciferase expression
originally detected in the brains of intact animals from the no. 887
line (Fig. 4) and most likely reflects an effect due to the site of
transgene insertion. Expression of the transgene in the liver was below
background and was not affected by estradiol treatment.
Synergistic activation of the oGR-9100LUC transgene by estradiol
and GnRH
Having established estradiol and GnRH responsiveness of 9,100 bp
of ovine promoter in transgenic mice, we next sought to determine if
the effect of these hormones was additive or synergistic on luciferase
expression. To address this issue, ovariectomized transgenic mice
derived from founder no. 884 receiving antiserum were treated with
estradiol and D-Ala-6-GnRH alone or in combination. Consistent with the
results of the first experiment, pituitary expression of luciferase was
approximately 9-fold higher in estradiol treated animals than that
observed in animals receiving D-Ala-6-GnRH (Fig. 6). When estradiol and D-Ala-6-GnRH were
administered in combination, pituitary luciferase activity was
approximately 2-fold higher than with estradiol alone, and nearly
20-fold higher than with D-Ala-6-GnRH treatment alone. Thus, the
combined estradiol and D-Ala-6-GnRH treatment increased pituitary
expression of the luciferase fusion gene to a level greater than the
sum of the individual hormone treatments, suggestive of synergistic
activation of the oGnRHR promoter by estradiol and GnRH. Although
significant (P < 0.05), the effect of estradiol on
luciferase expression in the brain was not as pronounced as that
observed in Fig. 4. Luciferase expression in the liver was below
background and not affected by treatments.
Elements conferring tissue-specific expression and hormonal
responsiveness are contained within 2700 bp of proximal 5' flanking
sequence from the ovine GnRHR gene
Despite the absence of in vitro activity, 9,100 bp of
oGnRHR 5' flanking sequence were sufficient for both tissue specific
expression and hormonal responsiveness in transgenic mice. To begin to
delimit the essential promoter regions, we constructed multiple lines
of transgenic mice harboring a fusion gene consisting of approximately
2,700 bp of 5' flanking sequence from the oGnRHR receptor gene linked
to the cDNA encoding luciferase. As with the -9,100 lines, luciferase
expression was primarily confined to the pituitary gland, brain, and
gonads of F1 progeny from three different founder animals (Fig. 7). It is, however, interesting to note
that the relative level of expression of the -2,700 construct was
consistently lower than the -9,100 construct. Finally, similar to the
-9,100 lines of transgenic mice, luciferase expression in the
pituitary gland was higher in female animals, while males generally
displayed higher gonadal expression.
|
. GnRH immunoneutralization reduced pituitary
expression of luciferase by greater than 90% and led to the expected
decrease in serum concentrations of LH (Fig. 8A). Treatment with estradiol increased
pituitary luciferase levels approximately 20-fold compared with
treatment with antiserum alone. Brain luciferase levels were unaffected
by treatment with AS but increased ap-proximately 2-fold by
estradiol treatment. Expression of luciferase in the liver was
below background regardless of treatment.
Based on the data in Fig. 8A, it appears that the element(s) conferring
estradiol responsiveness reside within 2,700 bp of proximal 5' flanking
region from the oGnRHR gene. Furthermore, the diminution of pituitary
expression of luciferase associated with antiserum treatment suggests
that the elements underlying GnRH responsiveness are also present in
the 2,700-bp promoter fragment. However, to confirm the latter, male
mice of the same transgenic line were castrated and treated with
antiserum alone or in combination with every other hour pulses of
D-Ala-6-GnRH. As with the ovariectomized females, treatment with
antiserum led to a significant reduction of transgene expression in the
pituitary gland and serum concentrations of LH (Fig. 8B). Replacement
with D-Ala-6-GnRH in the AS-treated animals restored both pituitary
luciferase and serum concentrations of LH to levels not different from
castrate controls. Expression of the transgene in the brain was not
affected by treatments.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit gene, the unique LH and FSH ß-subunit genes, and
the GnRHR gene. Furthermore, expression of each of these genes is
tightly regulated by a variety of different stimulatory and inhibitory
endocrine inputs. Of the different hormones that directly mediate
gonadotrope function, perhaps none are more fundamental than GnRH.
Quite simply, in the absence of GnRH input, gonadotrope function
ceases. Thus, the GnRHR represents the site that ultimately receives
and mediates the primary stimulatory input to gonadotropes. In the past
several years, we have identified central features of the murine GnRHR
gene that mediate hormonal responsiveness and cell-specific expression.
In regard to the latter, we have suggested that a tripartite enhancer
localized to less than 500 bp of 5' flanking sequence consisting of a
binding site for steroidogenic factor-1, a consensus AP-1 element, and
an element we have termed the GnRH receptor activating sequence (GRAS)
are sufficient for basal activity in the gonadotrope-derived T3–1
cell line (16, 20, 21). Similar mechanisms may underlie cell-specific
expression of the rat GnRHR gene (49). In contrast, although SF-1 may
contribute to activity of the human GnRHR gene promoter (50), the other
elements which comprise the tripartite enhancer of the murine GnRHR
gene promoter, are not found in either the human, ovine, or bovine
genes (17, 35, 36) (GenBank Accession No. AF034950). Thus, the
mechanisms underlying cell-specific expression of nonrodent GnRHR genes
remain undetermined.
Herein, we find that in stark contrast to both the murine and rat GnRHR
genes, greater than 9,000 bp of 5' flanking sequence from the ovine
GnRHR gene is devoid of functional activity in T3–1 and LßT2
cells. Our inability to detect activity of the ovine promoter by
transient expression in the murine gonadotrope-derived T3–1 and
LßT2 cell lines suggested at least two possibilities; 1) Either the
ovine promoter fragment lacked critical cis-acting elements; or 2) the
cell lines were lacking necessary transcription factors. The transgenic
mouse model has allowed us to eliminate the former. Specifically, the
ability of both the 9,100, and 2,700 bp promoters to direct luciferase
expression to established sites of GnRHR expression indicates that the
elements necessary to target tissue-specific expression are located
within 2,700 bp of 5' flanking sequence and that mouse pituitary cells
are capable of supporting transcriptional activity of the ovine GnRHR
gene promoter. Consequently, the simplest explanation for the
divergence in expression between the cell lines and transgenic mice is
that, unlike normal mouse gonadotropes, the gonadotrope-derived cell
lines used in the transfections either lack a necessary factor for
activation of the oGnRHR gene promoter or express a factor that
prevents activation of the oGnRHR gene promoter. Furthermore, the
divergent activity of the ovine promoter in cell lines vs.
transgenic mice underscores the importance of expanding in
vitro analyses of promoter function to in vivo
models—an emerging theme in recent studies of gene expression (40, 51, 52, 53). Finally, it is interesting to note that pituitary luciferase
expression was consistently higher in transgenic animals harboring
9,100 bp of 5' flanking sequence than those with 2,700 bp, suggesting
the presence of an upstream enhancer region located between -9,100 and
-2,700 bp of the oGnRHR gene promoter.
In addition to providing an avenue for studying tissue-specific expression, the use of transgenic mice permits assessment of promoter activity in a complex physiological setting that is difficult to fully recapitulate in vitro. Of particular interest to us are the mechanisms underlying GnRH and estradiol regulation of GnRHR gene expression. In our studies, we have used castrated male and female mice to eliminate gonadal input of steroid and peptide hormones. In addition, we have immunoneutralized GnRH in the transgenic mice (18). Thus, we can examine the effects of both estradiol and GnRH independent of endogenous input from these hormones. This model has allowed us to identify for the first time a gonadotrope-specific promoter that is directly activated by estradiol in the pituitary gland of transgenic mice.
Our data suggest that element(s) necessary for estradiol responsiveness are located within 2,700 bp of 5' flanking region of the oGnRHR gene. This stands in contrast to analysis of promoter function by transient expression assays in transformed cell lines in which we were unable to detect regulation by estradiol despite estradiol activation of a fusion gene consisting of tandem EREs linked to a heterologous, minimal promoter. Thus, responsiveness of the oGnRHR gene to estradiol may not be conferred by a canonical ERE. In support of this notion, computer analysis of 2,700 bp of the oGnRHR 5' flanking sequence, does not reveal an ERE (TESS analysis), nor has a canonical ERE been identified in any GnRHR gene examined (16, 17, 34, 35, 36). Furthermore, because none of the cell lines revealed estradiol regulation of any ovine promoter construct, there may be a fundamental problem with these in vitro models for studying estradiol regulation of the GnRHR gene. Consequently, transgenic mice may represent one of the few physiological models for defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.
Like estradiol responsiveness, element(s) conferring GnRH
responsiveness of the ovine GnRHR gene are also contained within 2,700
bp of proximal promoter. Similarly, 1,900 bp of 5'flanking sequence
from the murine GnRHR gene was responsive to GnRH, but not to
estradiol, in transgenic mice (18). Analysis of the GnRH response of
the murine promoter in transient transfection of T3–1 cells has
pinpointed the AP-1 element as the primary target for GnRH stimulation
via activation of protein kinase C (22, 23). At issue is whether a
similar mechanism accounts for GnRH regulation of the ovine GnRHR gene.
Unfortunately, we are unable to detect GnRH responsiveness of the ovine
promoter in either T3–1 or LßT2 cells (data not shown). Thus, as
with estradiol regulation, definition of the elements and factors that
mediate GnRH responsiveness of nonrodent GnRHR genes may require the
use of transgenic mice. It is interesting to note that GnRH induces a
fos response in the ovine pituitary (54); however, the
GnRH-responsive AP-1 element defined in the murine GnRHR gene (22, 23)
is not conserved in the 2700-bp ovine promoter, suggesting that
different mechanisms may underlie GnRH regulation of the GnRHR gene in
nonrodent species.
To attempt to mimic the endocrine changes associated with the preovulatory LH surge, we treated castrated mice injected with antiserum with estradiol and D-Ala-6-GnRH in combination. We found that these hormones in combination had a 2-fold and 20-fold greater stimulatory effect than either estradiol or GnRH alone, respectively. This synergistic effect may be mediated at response elements located in the promoter or due to heightened pituitary sensitivity to D-Ala-6-GnRH resulting from estradiol induction of endogenous GnRH receptors. Regardless of the precise mechanism, these data establish the ability of estradiol and GnRH to jointly enhance GnRHR gene expression and thus underscores the established role of these hormones in potentiating pituitary sensitivity to GnRH during the preovulatory period. Finally, since the 2,700-bp promoter retained both GnRH and estradiol responsiveness, it seems likely that the synergistic effects of these two hormones is also retained. Nevertheless, as we have only directly tested this in mice harboring the 9,100-bp promoter, we cannot eliminate the possibility that elements residing upstream of 2,700 bp may contribute to the synergistic response observed with the combined GnRH and estradiol treatment.
In summary, despite the absence of functional activity of the oGnRHR gene promoter in vitro, approximately 2,700 bp of proximal promoter from the oGnRHR gene contain element(s) necessary to direct tissue-specific expression as well as responsiveness to both estradiol and GnRH in transgenic mice. These data underscore the utility of transgenic mice as an adjunct for analyzing endocrine regulation of gene expression. Finally, although the ability of estradiol to increase pituitary sensitivity to GnRH has been established for more than 20 yr, the underlying mechanisms have remained undefined. This report is the first demonstration of estradiol regulation of transcription of the GnRHR gene directly at the level of the pituitary gland and defines a paradigm for elucidating the mechanisms underlying estradiol regulation of GnRHR gene expression.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Supported by NIH Individual NRSA F32-HD-08169.
Received September 20, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. Xie, K. H. Allen, A. Marguet, K. A. Berghorn, S. P. Bliss, A. M. Navratil, J. L. Guan, and M. S. Roberson Analysis of the Calcium-Dependent Regulation of Proline-Rich Tyrosine Kinase 2 by Gonadotropin-Releasing Hormone Mol. Endocrinol., October 1, 2008; 22(10): 2322 - 2335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kowase, H. E. Walsh, D. S. Darling, and M. A. Shupnik Estrogen Enhances Gonadotropin-Releasing Hormone-Stimulated Transcription of the Luteinizing Hormone Subunit Promoters via Altered Expression of Stimulatory and Suppressive Transcription Factors Endocrinology, December 1, 2007; 148(12): 6083 - 6091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T Zhang and M S Roberson Role of MAP kinase phosphatases in GnRH-dependent activation of MAP kinases J. Mol. Endocrinol., February 1, 2006; 36(1): 41 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xie, S. P. Bliss, T. M. Nett, B. J. Ebersole, S. C. Sealfon, and M. S. Roberson Transcript Profiling of Immediate Early Genes Reveals a Unique Role for Activating Transcription Factor 3 in Mediating Activation of the Glycoprotein Hormone {alpha}-Subunit Promoter by Gonadotropin-Releasing Hormone Mol. Endocrinol., October 1, 2005; 19(10): 2624 - 2638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-Y. Kam, K.-H. Jeong, E. R. Norwitz, E. M. Jorgensen, and U. B. Kaiser Oct-1 and Nuclear Factor Y Bind to the SURG-1 Element to Direct Basal and Gonadotropin-Releasing Hormone (GnRH)-Stimulated Mouse GnRH Receptor Gene Transcription Mol. Endocrinol., January 1, 2005; 19(1): 148 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Granger, V. Ngo-Muller, C. Bleux, C. Guigon, H. Pincas, S. Magre, D. Daegelen, A. Tixier-Vidal, R. Counis, and J.-N. Laverriere The Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene Directs the Expression of the Human Placental Alkaline Phosphatase Reporter Gene in Gonadotrope Cells in the Anterior Pituitary Gland as well as in Multiple Extrapituitary Tissues Endocrinology, February 1, 2004; 145(2): 983 - 993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. Cheng, B. K. C. Chow, and P. C. K. Leung An Activator Protein 1-Like Motif Mediates 17{beta}-Estradiol Repression of Gonadotropin-Releasing Hormone Receptor Promoter via an Estrogen Receptor {alpha}-Dependent Mechanism in Ovarian and Breast Cancer Cells Mol. Endocrinol., December 1, 2003; 17(12): 2613 - 2629. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, J. Hicks, P. Khanbolooki, S.-J. Kim, C. Yan, Y. Wang, and D. Boyd Transgenic Mice Demonstrate Novel Promoter Regions for Tissue-Specific Expression of the Urokinase Receptor Gene Am. J. Pathol., August 1, 2003; 163(2): 453 - 464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Ellsworth, B. R. White, A. T. Burns, B. D. Cherrington, A. M. Otis, and C. M. Clay c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in {alpha}T3-1 Cells Endocrinology, March 1, 2003; 144(3): 839 - 849. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
