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(ER)- and ERß-Expressing GT1–7 GnRH Neurons1
Institute for Medical Sciences (D.R., D.D.B.) and the Department of Physiology (D.D.B.), University of Toronto, and Division of Reproductive Science, Toronto Hospital Research Institute, Toronto, Ontario, Canada M5G 2C4
Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology/Division of Reproductive Science, University of Toronto/Toronto Hospital Research Institute, 200 Elizabeth Street, CCRW 3–832, Toronto, Ontario, Canada M5G 2C4. E-mail: d.belsham{at}utoronto.ca
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Abstract |
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(ER) and the recently described ERß
mRNAs. We also detected the presence of both receptor subtypes in the
GT1–7 neurons by Western blot analysis using specific ER antibodies.
By Northern blot analysis of total GT1–7 RNA, we found that
17ß-estradiol (1 nM) down-regulates GnRH mRNA levels to
approximately 55% of basal levels over a 48-h time course. This effect
appears to occur specifically through an ER-mediated mechanism, as ICI
182,780, a complete ER antagonist, blocks the repression of GnRH mRNA
levels by estradiol. The recently reported ER-specific
agonist/ERß-specific antagonist
2,2-bis-(p-hydroxyphenyl-1,1,1-trichloroethane (HPTE), a
methoxychlor metabolite, also down-regulated GnRH gene expression. The
repression of GnRH mRNA levels appears to occur at the transcriptional
level, as simian virus 40 T antigen mRNA expression, which is under the
control of 2.3 kb of the rat GnRH 5'-regulatory region, mimics the
down-regulation of GnRH after treatment with estradiol. As the rat GnRH
regulatory region in GT1–7 neurons does not appear to harbor a classic
estrogen response element, the mechanism involved in the repression of
GnRH has yet to be determined. These results suggest that estradiol
directly regulates GnRH gene expression at the level of the GnRH neuron
and may exert its neuroendocrine control through direct interaction
with specific receptors expressed in these cells. |
Introduction |
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Due to the dramatically divergent results observed, it is still controversial whether estrogen modulates GnRH transcription in vivo (2). Conflicting results have been obtained depending on the time the rats were killed, the location of the GnRH neuron within the medial septal-preoptic hypothalamic regions, the length of time after castration, and the dose and time of estrogen treatment (3). It has been postulated that stimulation of GnRH gene expression occurs via the same neurotransmitter pathways found to be responsible for the enhancement of GnRH secretion (3). On the other hand, there is almost no information available regarding the mechanisms of negative regulation of GnRH gene expression by estrogen.
Estrogen may influence the GnRH neuron directly, but currently there is little evidence for the expression of estrogen receptors (ERs) in native GnRH neurons. In the early 1980s, the estrogen responsiveness of the GnRH neuron was established electrophysiologically by Kelley et al. (4), who demonstrated that estrogen inhibits the firing of GnRH neurons in hypothalamic slices obtained from female guinea pigs. At almost that same time, however, Shivers et al. reported that GnRH neurons do not concentrate estradiol (5). This study was followed by a number of reports using immunocytochemistry performed in rats (6), guinea pigs (7), sheep (8), and monkeys (9), in which none or only a few rare GnRH-immunoreactive hypothalamic neurons colocalized with ER expression, which led to the present belief that GnRH neurons could not be directly affected by estrogen. Instead, estrogen-receptive interneurons contacting GnRH neurons are thought to be responsible for mediating the effects of estrogen and other sex steroids on GnRH synthesis and secretion (5, 10, 11).
The scarcity of GnRH neurosecretory neurons makes study of their cell and molecular biology difficult. Classical in vivo approaches cannot establish the direct action of agents such as gonadal steroids on the GnRH neuron or on GnRH transcription, mainly because the GnRH system receives input from other steroid-sensitive cells. In an attempt to produce a suitable model to study the GnRH gene, a targeted tumorigenesis technique was used to develop a murine immortal cell line of GnRH-secreting hypothalamic neurons (GT1 cells) (12). To date, the GT1 cells have proven to be the best characterized cell model available to study biology of the hypothalamic GnRH neurons (13, 14).
The development of the GT1 cell line has allowed study of the direct
actions of sex steroids on GnRH neurons. Glucocorticoid
receptor-mediated repression of GnRH promoter activity has been
demonstrated in GT1 cells (15). Poletti et al. reported that
GT1 neurons also bind androgens and metabolize this gonadal steroid via
the two major enzymatic pathways involved in the modulation of androgen
action (16, 17). In agreement with these initial studies, our
laboratory has demonstrated the expression of a functional androgen
receptor in the GT1 cells and down-regulation of GnRH messenger RNA
(mRNA) levels after 5-dihydrotestosterone treatment (18). In
addition to androgen-binding activity, Poletti et al.
detected the presence of specific binding sites for estrogen in GT1
cells (16). Another recent study has demonstrated that these cells
express ER mRNA and exhibit specific estrogen-binding activity, and
through transient transfection analysis found that the ER expressed in
the GT1 cells can activate an exogenous estrogen-responsive element
(ERE)-luciferase plasmid (19). These findings strengthened the
hypothesis that estrogen may directly regulate gene expression in the
GnRH neuron.
To determine whether regulation of gene expression by estrogen
contributes to its negative feedback action on GnRH neurons, we studied
the direct effect of estrogen on GnRH mRNA levels in GT1–7 neurons. We
report both ER and ERß mRNA and protein expression in GT1–7
GnRH-secreting neurons. We also demonstrate that 17ß-estradiol as
well as 2,2-bis-(p-hydroxyphenyl-1,1,1-trichloroethane
(HPTE), an ER agonist, down-regulate GnRH mRNA levels in GT1 cells
through an ER-specific mechanism. Interestingly, the down-regulatory
effect appears to be mediated at the transcriptional level. These
observations provide evidence that the estrogen-mediated inhibition of
GnRH gene expression is exerted via ERs in the GT1–7 cells, the
current model system of the hypothalamic GnRH neuron.
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Materials and Methods |
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Northern blot analysis
Northern blot analysis was performed as previously described
(21). Briefly, total cellular RNA was isolated by the guanidinium
thiocyanate phenol chloroform extraction method (22). Ten micrograms of
total RNA were size fractionated in 1% agarose-formaldehyde gels and
transferred to GeneScreen membranes (NEN Life Science Products, Boston, MA) by capillary blotting (23). The filters
were hybridized with rat GnRH complementary DNA (cDNA) (24) or simian
virus 40 (SV40) T antigen (TAg) cDNA (25). Hybridization with mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA generated by
RT-PCR or human 
-actin cDNA (26) was used to control for variations
in gel loading and transfer efficiency. GnRH and -actin probes were
routinely hybridized concurrently to minimize error caused by stripping
and reprobing of the membrane. Prehybridization for 2–16 h and
hybridization for 16 h were conducted in a 25% formamide
hybridization buffer [1% wt/vol BSA, 1 mM EDTA, 0.5
M Na2HPO4, 5% (wt/vol) SDS, and
25% formamide] at 55 C. The cDNA probes were labeled using random
hexamers and [32P]deoxy-ATP (6000 Ci/mmol; NEN Life Science Products) incorporated with the Klenow fragment of DNA
polymerase I (27). Blots were washed at high stringency (55 C; 0.5
x SSC, 0.1% SDS) and exposed to Fuji film (obtained from
Fisher Scientific Ltd., Nepean, Ontario, Canada) at -70 C
with intensifying screens for 4–48 h. Autoradiographs were scanned
with a Hewlett-Packard Co. ScanJet 3p Scanner (Palo Alto,
CA), and GnRH, TAg, actin, and GAPDH mRNA signals were quantified by
densitometry using the NIH Image program. Statistical significance of
the results was determined by Student’s t test, as
indicated.
RT-PCR and DNA sequencing
Total RNA was isolated from GT1–7 cells and from mouse ovary,
cerebellum, and testes, as described above. First strand cDNA was
synthesized from 1–10 µg deoxyribonuclease I-treated RNA, using
SuperScript II reverse transcriptase (RT) and oligo(deoxythymidine), as
described in the Superscript II cDNA Synthesis Kit (Life Technologies, Inc.). The specificity of each amplification
reaction was monitored in control reactions, where amplification was
carried out on samples in which the RT was omitted. Amplification of H3
histone cDNA (222 bp) was used as a control to determine first strand
cDNA quality.
PCR amplifications were performed with 1.25 U Taq polymerase
(Life Technologies, Inc.) in a 50-µl reaction for 35
cycles (1.5 min at 94 C, 1.5 min at 57 C for ER and 65 C for ERß,
and 1.5 min at 72 C). Primers were designed from the mouse ER sequences
in GenBank to span introns. External and internal primers were used to
amplify ER cDNA. A PCR product of the expected size (344 bp) was
amplified with the external primers 5'-GAATTCAATTCTGACAATCGACGCCAG-3'
and 5'-GAATTCGTGCTTCAACATTCTCCCTCC-3'. This external PCR product was
used as a template for a second PCR using the internal primers
5'-GAATTCGAGAAAGGAAACATGATCATG-3' and GAATTCTTCATCATGCCCACTTCGTAA-3' to
amplify a product of 247 bp. ERß cDNA was amplified using external
primers for ERß (5'-GAATTCTAGCCACCCACTGCCAATCAT-3' and
5'-GAATTCCACACCTTTCTCTCCTGGATC-3'; expected size, 407 bp). These
products were separated by 1.8% agarose gel electrophoresis, Southern
transferred onto GeneScreen hybridization membrane (NEN Life Science Products) by capillary blotting (23), and hybridized
with an internal ERß cDNA probe, using the hybridization and washing
conditions described above. The internal ERß probe was generated from
mouse ovarian cDNA using primers 5'-GAATTCCAGAACCTCAAAAGAGTCCTT-3' and
5'-GAATTCCGTAACACTTGCGAAGTCGGC-3', and its identity was confirmed by
sequencing. Primers used to amplify the rat GAPDH cDNA were sense
5'-TCCACCACCCTGTTGCTGTAG-3' and antisense 5'-GACCACAGTCCATGACATCACT-3'.
All primers were made by ACGT Corp. (Toronto, Canada).
ER and ERß PCR products from the GT1–7 cells were electrophoresed
in 1.8% agarose gels, stained with 1 µg/ml ethidium bromide, and
visualized under UV light. Corresponding DNA fragments were isolated
and purified using GeneClean (BIO 101, Inc., Vista, CA) and subcloned
into pCR2.1-TOPO cloning vector using the TOPO TA Cloning Kit
(Invitrogen, Carlsbad, CA). DNA was prepared for
sequencing using the Wizard Plus Miniprep Kit (Promega Corp., Madison, WI) according to the manufacturer’s
instructions. DNA sequencing was performed by the dideoxy-sequencing
method of Sanger et al. (28) using the T7 Sequencing Kit
(Pharmacia Biotech, Piscataway, NJ) and
[35S]deoxy-ATP (10 mCi/ml; Amersham Pharmacia Biotech, Arlington Heights, IL). Sequencing reactions were run
on 6% polyacrylamide gels for 3–6 h. Gels were then dried and exposed
to Kodak X-600 autoradiography film (Eastman Kodak Co., Rochester, NY) for 16 h. Sequences were confirmed
using the NIH Blast Program.
Immunoprecipitation and Western blot analysis
Confluent GT1–7 cells were washed twice with ice-cold PBS
containing protease inhibitors leupeptin (10 µg/ml), aprotinin (10
µg/ml), and PMSF (1 mM) and lysed with Nonidet P-40 lysis
buffer [50 mM HEPES (pH 7.25), 150 mM NaCl,
0.1 mM ZnCl2, 2 mM EDTA, 1%
(vol/vol) Nonidet P-40, and 2 mM PMSF]. Mouse ovary,
testes, cerebellum, or hypothalami (controls) were homogenized in
Nonidet P-40 lysis buffer. Cell debris was removed by centrifugation at
12,000 rpm for 10 min at 4 C, and the supernatants were used for
immunoprecipitation and/or Western blotting analysis. For the ERß
immunoprecipitation, the clarified supernatant was incubated with 4%
(vol/vol) Protein G-Plus agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C, then centrifuged at
12,000 rpm for 10 min to remove the agarose beads. ERß was
immunoprecipitated from the precleared supernatant by the addition of 1
µg/ml polyclonal rabbit antirat ERß antisera and 4% (vol/vol)
Protein G-Plus agarose overnight with agitation at 4 C. Samples
were centrifuged at 12,000 rpm for 10 min at 4 C, and the pellets were
washed four times with Nonidet P-40 lysis buffer. Crude protein
extracts (for ER) or immunoprecipitated proteins (for ERß; 50 µg
of each) were suspended in Laemmli sample buffer to a final
concentration of 1 x and boiled for 5 min before resolution on a
10% SDS-PAGE gel. Proteins were transferred to an Immobilon
polyvinylidene difluoride membrane (Millipore Corp.,
Bedford, MA). The membranes were incubated in TBST blocking solution
[50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.2%
(vol/vol) Tween-20, and 5% powdered skim milk] for 24 h with 1
µg/ml of either polyclonal rabbit antimouse ER or antirat ERß
antisera [ER, MC-20 (directed toward the carboxyl-terminal of the
mouse ER; sc-542, Santa Cruz Biotechnology, Inc.);
ERß, PA1–310 (Affinity BioReagents, Inc., Golden,
CO)]. For peptide blocking analysis, the ER or ERß immunizing
peptide [5 µg/ml; MC-20 blocking peptide (Santa Cruz Biotechnology, Inc.) or PA1.1–310 (Affinity BioReagents, Inc.), respectively] was preincubated with their respective
antibodies for 1 h at room temperature before
immunoprecipitation and/or Western blot analysis. Immunoreactive
bands were visualized with horseradish peroxidase-labeled secondary
goat antirabbit antiserum at a 1:5000 dilution and enhanced
chemiluminescence (ECL Kit, Amersham Pharmacia Biotech,
Inc., Oakville, Canada) as described by the manufacturer.
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Results |
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(19). We have used RT-PCR to
test for the presence of cDNAs from both ER and ERß in GT1–7
cells. Our results confirm that ER is expressed in GT1–7 cells,
but, additionally, ERß transcripts are present in these cells (Fig. 1
, A and 1B). Mouse ovary cDNA was
amplified using the ER primers and served as a positive control for
both ER mRNAs. DNA sequencing of the cloned PCR products confirmed
their identity with the mouse ER sequences. The ERß cDNA was
consistently difficult to amplify by PCR in both GT1 cells and mouse
ovary, suggesting either low expression levels or an inherently
unstable ERß mRNA.
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antiserum recognizes an immunoreactive band at 67 kDa in GT1–7 cells
(Fig. 2A), which comigrates with a
protein of identical mobility highly expressed in mouse ovary and to a
lower level in mouse testes, as expected from analysis of ER
transcript distribution in the rat (29). This protein is not expressed
in mouse cerebellum, which, in the rat, was previously found to express
mainly ERß (29). Although we have tried a number of commercially
available anti-ER antiserum, none was able to detect only a single
band at 67 kDa, the predicted molecular mass of ER, in the GT1–7
cells or the other tissues tested. We often found a few smaller
immunoreactive bands on the Western blots. Interestingly, all of them
were specifically competed by the ER peptide, suggesting that the
smaller bands are probably degradation products or alternatively
truncated ER products.
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). These complexes were also present in mouse
ovary and hypothalamus, as expected from the analysis of transcript
distribution of ERß in the rat (29). The 55-kDa band, which
corresponds to the predicted molecular mass, was specifically competed
by the immunizing peptide. Interestingly, the closely migrating bands
at approximately 80 kDa were also competed, although to a lesser
degree, by the ERß peptide. We presently have no explanation for the
larger band, but it may simply represent a nonspecific reaction to the
antiserum. Our results confirm the previous study, indicating the
presence of ER in the GT1 cells, but to our knowledge, this is the
first report indicating ERß expression, at both mRNA and protein
levels, in GT1–7 neurons.
Effect of 17ß-estradiol on GnRH mRNA levels
To understand the potential role of ERs in the GnRH neuron, we
looked for a direct effect of estrogen on these cells. The GT1 cells
are the optimal system to study the direct effects of estrogen on GnRH
gene expression due to the homogeneity of the cell population. These
studies are a difficult, if not a technically impractical, task
in vivo. We used the GT1–7 cells exposed to 17ß-estradiol
over a 48-h time course to study the effect of estrogen on GnRH mRNA
synthesis. Repression of GnRH mRNA levels was statistically significant
(P < 0.005) over a 48-h time course upon treatment
with 1 nM 17ß-estradiol (Fig. 3, A and B). The medium was devoid of
phenol red, which was previously shown to exhibit estrogenic effects on
the proliferation of MCF-7 human breast cancer cells (30), although we
found that there was little if any difference in the level of
repression using either phenol red-devoid medium or medium with phenol
red. Vehicle alone (14 nM dimethylsulfoxide) did not change
GnRH mRNA levels over time, although there was a negligible increase
(not statistically significant) in GnRH mRNA levels compared with that
in nonvehicle controls (data not shown). When the initial studies were
conducted, we used -actin as a loading control. Actin has been shown
to be regulated by estradiol during mitogenic stimulation of rat
uterine cells (31); therefore, we also probed these same blots with
mouse GAPDH and 18S cDNAs. All three probes exhibited a similar
hybridization pattern, indicating that in the GT1–7 cells, -actin
gene expression is not regulated by estradiol over a 48-h time course,
suggesting that it is an appropriate loading control.
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). ICI 182,780 alone did not
significantly change steady state GnRH mRNA levels. This indicates that
repression of GnRH mRNA levels by estradiol is an ER-mediated effect,
but as ICI 182,780 antagonizes both ER and ERß activities, we
cannot yet determine which ER is involved in this process.
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and an antagonist for ERß in a
number of different cell lines, including HepG2 cells, and on numerous
estrogen-sensitive exogenous promoters (32). We treated the GT1–7
neurons with HPTE to decipher which ER may be responsible for the
down-regulation of GnRH mRNA levels by estradiol. At 24 and 36 h,
100 nM HPTE mimics the effects of estradiol in GT1–7 cell
culture experiments. HPTE, either alone or in combination with
estradiol, down-regulates GnRH mRNA levels to approximately 50% of
basal GnRH mRNA levels (Fig. 4B). These results indicate that ER is
involved in the regulation of GnRH synthesis by estradiol, but we
cannot yet determine whether the two ERs form a heterodimer complex to
achieve the repression of GnRH mRNA levels. It has been shown that
hydroxytamoxifen (OHT), an antagonist of ER, exhibits differential
binding activity to either ER isoforms, with an apparent shift in the
dose response toward the lower concentrations for ERß compared with
ER (33). Using this information, we treated the GT1–7 cells with
OHT to distinguish ER isoform activities at different concentrations.
Unfortunately, in our hands, OHT at concentrations between
10-9–10-7 M appears to exhibit
agonist activity, similar to that of estradiol, in GT1–7 cells and
therefore could not be used in these studies. Until a more useful agent
is described to distinguish between the activities of the two ER
isoforms, we can report only that ER is necessary for the repression
of GnRH mRNA levels. We continue to look for evidence that ERß is
also involved in this process in the GnRH-secreting GT1–7 cell
line.
Transcriptional regulation of GnRH mRNA levels by
17ß-estradiol
The down-regulation of GnRH synthesis by estradiol in the GT1–7
neurons may be achieved either at the transcriptional level or by
changes in RNA stability. To understand how estradiol down-regulates
GnRH steady state mRNA levels, we studied changes in the mRNA levels of
the exogenous oncogene expressed in the immortalized GT1–7 cell line.
The GT1 cells were produced using the potent oncogene, TAg, under the
control of 2.3 kb of the rat GnRH 5'-regulatory region. There is no
endogenous expression of this protein in these cells. Two TAg
transcripts are produced through alternative splicing at 2450 and 2200
bp, although the expression of the larger transcript is low in GT1–7
cells (12). We have used TAg cDNA to probe the same Northern blots used
to detect the down-regulation of GnRH mRNA by estradiol. TAg mRNA
expression mimics that of GnRH (Fig. 5).
This indicates that the region responsive to estrogen, probably through
an ER homo- or heterodimer, lies within the same region used to create
the cell line. This region encompasses DNA from -2987 to -1172 and
from -441 to +104 of the rat GnRH 5'-regulatory region (12). Although
this region does not appear to contain a classic ERE, it is possible
that the down-regulatory effect mediated by estrogen at the
transcriptional level uses a unique ERE sequence.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renewed interest in the mechanism of estrogen action on the GnRH neuron
has been generated by a few studies using the model GT1 cell line,
which have provided new evidence suggesting that functional ERs may be
expressed in the hypothalamic GnRH neuron. These studies have indicated
that the GnRH-secreting GT1 neuronal cell line contains high affinity
binding sites for estrogens (16), although as might have been
anticipated from previous in vivo studies of GnRH neurons,
the number of estradiol-binding sites found in GT1 cells appears to be
low (16, 19). Using sensitive RT-PCR, we have found that both ER and
ERß mRNAs are expressed in the GT1–7 cells. These findings confirm
the results from previous studies that indicated the presence of ER
transcripts in GT1 cells (19) and from a preliminary study that
indicated the expression of both ERs (34). We also confirmed that the
ER mRNAs are translated into their respective proteins using Western
analysis with specific ER or ERß antibodies.
The reason for the apparent absence of estrogen and other steroid receptors is probably due to the nature of the GnRH neuron itself and/or to limitations in the sensitivity of detection methods used at the time. GnRH neurons are rare and dispersed in the hypothalamus. Furthermore, it is possible that only subgroups of GnRH neurons contain steroid receptors, as noted with progestin receptors (35). Thus, classical approaches may not have been sensitive enough to detect very low levels of receptors within GnRH neurons. Furthermore, the reproductive status of the animal, ovariectomized or at various stages of the estrous cycle, may change the steroid responsiveness of the GnRH neuron. The sex steroid environment of the GnRH neuron may dictate the level of ER expressed, as autoregulation of ER gene expression is common (36, 37, 38). Similarly, there has been some variability in the detection of steroid receptor expression in the GT1–7 cell line, which may also be due to the steroid environment of the cells. Receptor mRNA levels may depend upon the type of serum used or the length of time the cells have been growing in the medium before cDNA preparation.
The in vivo studies examining the effects of estrogen on
hypothalamic GnRH mRNA levels have mostly been performed in the female
rat and have unfortunately led to a great deal of controversy. The GnRH
transcript levels have been analyzed in relation to the estrous cycle,
ovariectomy, and estrogen replacement (3, 39). The controversy
regarding either stimulatory or down-regulatory effects of estrogen
appear to result from the time of day the samples were taken and the
precise location of the brain in which GnRH transcript levels were
measured (40, 41). Peterson et al. were also able to
demonstrate that estrogen-induced increases in GnRH mRNA were due not
only to increased gene transcription, but also to increased mRNA
stability (41). As reviewed by Herbison (2), it appears that estrogen
has stimulatory effects on GnRH mRNA expression in only rostral
preoptic neurons and that a gradual increase in GnRH mRNA content, from
the low levels found in the morning in estrogen-treated rats, results
in a peak of expression in the early afternoon, before the onset of the
LH surge. The measurement of estrogen effects on GnRH mRNA synthesis in
estrogen-treated rats that were killed in the morning, a time when
estrogen exerts an inhibitory influence on LH secretion, is probably
responsible for the inhibitory effects on GnRH mRNA levels reported
(42, 43). Therefore, the current consensus appears to be that estrogen
at low levels inhibits GnRH mRNA expression relative to that in
ovariectomized rats, whereas higher concentrations, in association with
a circadian rhythm, enhance GnRH mRNA levels (2). Nevertheless, it is
difficult to dissect the direct vs. indirect action of
estrogen from in vivo studies. After exposing GT1–7 cells
to a low dose of 17ß-estradiol over 48 h, we found a significant
repression of GnRH mRNA levels. This effect on GnRH mRNA synthesis was
mimicked by the ER agonist/ERß antagonist HPTE, thus suggesting
that ER is involved in the regulation of GnRH synthesis by estradiol
in GT1–7 cells.
It has recently been demonstrated that ER and ERß may form
heterodimers that bind to DNA with an affinity similar to that of ER
homodimers and greater than that of ERß homodimers (44, 45).
Heterodimers have been shown to be capable of binding to steroid
receptor coactivator-1 when bound to DNA and can stimulate
transcription of a reporter gene in transfected cells (46). This
indicates that ER and ERß may cooperate in regulating
estrogen-responsive gene expression in cells expressing both ERs. We
have demonstrated that the GT1–7 neurons express both subtypes of the
ER. Our results showing HPTE repression of GnRH mRNA levels indicate
that ER is involved in the regulation of GnRH by estradiol, but we
cannot yet exclude the possibility that the two subtypes of ER form a
heterodimer complex to achieve the observed repression of GnRH mRNA
levels.
Several reports have shown that the activity of the 5'-regulatory
region of the GnRH gene is sensitive to steroid hormone-mediated
regulation. Using transient transfection experiments in human placental
cells, a stimulatory ERE has been localized to the 5'-flanking region
of the human GnRH gene (47). Examination of the rat GnRH promoter
transiently transfected into JEG-3 placental cells has identified
regions other than canonical EREs important for negative
transcriptional regulation of the rat GnRH gene by estrogen (48). To
date, only one study has reported estrogen-mediated negative regulation
of rat GnRH promoter activity in GT1 cells (49). Because no ER mRNA
expression was detected by RT-PCR in that study, cotransfection of an
ER expression vector was necessary to analyze the region responsible
for negative regulation of GnRH reporter constructs by estrogen. The
estrogen-responsive region was found to lie between -171 and -126;
however, direct binding of the ER to the DNA was not detected (49).
Although there is in vivo evidence of progestin receptor
expression in GnRH neurons (35), progesterone receptors have only been
reported in GT1 cells in a preliminary study (34). Despite this, the
direct binding of progesterone receptor to nonconsensus DNA sequences
of the rat GnRH promoter has been shown to repress rat GnRH gene
transcription in GT1 cells in the presence of progesterone (50). These
studies set a precedent for a nonclassical steroid hormone mode of
action in the GT1 neurons.
It is not clearly understood how estrogen represses GnRH mRNA
expression. The regulation of GnRH synthesis by estrogen in GnRH
neurons may occur at a variety of levels including the transcriptional
level, through changes in RNA stability, or a combination of both. It
has been established previously by transient transfection experiments
that rat (48) and human (47) GnRH promoter activities are sensitive to
estrogen-mediated regulation. Both of these studies relied on the
transfection of ER into the cells, thereby overlooking any potential
effect of ERß on the negative regulation of GnRH. We have examined
whether the steroid effects of estrogen on GnRH are exerted directly at
the level of the rat GnRH gene itself by measuring the expression
levels of TAg mRNA after estradiol treatment in GT1 cells. The GT1
cells were produced using the coding region of the TAg oncogene, under
the control of 2.3 kb of the rat GnRH 5'-regulatory region. We observed
down-regulation of TAg mRNA levels in a pattern that mimicked GnRH mRNA
down-regulation by estradiol. This result suggests that estrogen
mediates its control of the GnRH gene at the transcriptional level in a
region that is located within the 2.3-kb rat GnRH 5'-regulatory
sequence used to create the GT1–7 cell line. The 2.3-kb fragment
encompasses DNA from -2987 to -1172 and -441 to +104 of the rat GnRH
5'-regulatory region (12). Although this region of the rat GnRH gene
appears to lack the canonical ERE consensus sequence, it is entirely
possible that estrogen mediates its transcriptional regulation of GnRH
mRNA through either ER homo- or heterodimers that bind to a unique ERE
sequence or through an indirect mechanism via protein-protein
interactions.
Our study suggests that hypothalamic GnRH neurons may have the capacity
to be directly regulated by estrogen. Using the hypothalamic-derived
neuronal cell line, GT1–7, we have shown that steady state GnRH mRNA
levels are significantly down-regulated upon treatment with estradiol,
through an ER-mediated mechanism. The environmental estrogen HPTE
mimics the repression of GnRH gene expression by estradiol, which
implies that ER is involved in this effect. However, one must also
consider that the GT1–7 cell line represents a single population of
neurons outside of its natural environment and cell contacts, and thus
may not respond to estrogen in the same manner as a GnRH neuron
in situ. Nevertheless, we have found that ERs of both
subtypes are expressed within GT1–7 cells. Furthermore, our data
indicate that stimulation of the GT1–7 neuron by estrogen is
ultimately realized at the level of the GnRH 5'-regulatory region.
Taken together, these results suggest that estrogen could very well
contribute directly to the physiology of the GnRH neuron and that
further investigation of direct effects of estrogen on GnRH neuronal
function in vivo is warranted.
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Acknowledgments |
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Footnotes |
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2 Medical Research Council of Canada Scholar and a Canada Foundation
for Innovation Researcher.
Received March 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone in GnRH-secreting GT1–7 hypothalamic neurons.
Endocrinology 139:1108–1114 cells. Neuron 1:605–613[CrossRef][Medline]
and
ß. Endocrinology 138:863–870, and ß.
Program of the 80th Annual Meeting of the Endocrine Society, New
Orleans, LA, p 71 (Abstract)
, and -ß, progesterone and androgen
receptors in immortalized GnRH-secreting neurons. Program of the 80th
Annual Meeting of the Endocrine Society, New Orleans, LA, p 444
(Abstract)
. Mol Endocrinol 11:1486–1496This article has been cited by other articles:
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  H. Otani, F. Otsuka, M. Takeda, T. Mukai, T. Terasaka, T. Miyoshi, K. Inagaki, J. Suzuki, T. Ogura, M. A Lawson, et al. Regulation of GNRH production by estrogen and bone morphogenetic proteins in GT1-7 hypothalamic cells J. Endocrinol., October 1, 2009; 203(1): 87 - 97. [Abstract] [Full Text] [PDF]
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 Z. Chu, J. Andrade, M. A. Shupnik, and S. M. Moenter Differential Regulation of Gonadotropin-Releasing Hormone Neuron Activity and Membrane Properties by Acutely Applied Estradiol: Dependence on Dose and Estrogen Receptor Subtype J. Neurosci., April 29, 2009; 29(17): 5616 - 5627. [Abstract] [Full Text] [PDF]
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 C. A. Christian, C. Glidewell-Kenney, J. L. Jameson, and S. M. Moenter Classical Estrogen Receptor {alpha} Signaling Mediates Negative and Positive Feedback on Gonadotropin-Releasing Hormone Neuron Firing Endocrinology, November 1, 2008; 149(11): 5328 - 5334. [Abstract] [Full Text] [PDF]
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 L. Hu, R. L. Gustofson, H. Feng, P. Ki Leung, N. Mores, L. Z. Krsmanovic, and K. J. Catt Converse Regulatory Functions of Estrogen Receptor-{alpha} and -{beta} Subtypes Expressed in Hypothalamic Gonadotropin-Releasing Hormone Neurons Mol. Endocrinol., October 1, 2008; 22(10): 2250 - 2259. [Abstract] [Full Text] [PDF]
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 W. E. Ottowitz, D. D. Dougherty, A. J. Fischman, and J. E. Hall [18F]2-Fluoro-2-Deoxy-D-Glucose Positron Emission Tomography Demonstration of Estrogen Negative and Positive Feedback on Luteinizing Hormone Secretion in Women J. Clin. Endocrinol. Metab., August 1, 2008; 93(8): 3208 - 3214. [Abstract] [Full Text] [PDF]
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J. Pielecka-Fortuna, Z. Chu, and S. M. Moenter Kisspeptin Acts Directly and Indirectly to Increase Gonadotropin-Releasing Hormone Neuron Activity and Its Effects Are Modulated by Estradiol Endocrinology, April 1, 2008; 149(4): 1979 - 1986. [Abstract] [Full Text] [PDF]
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H. Abe, K. L. Keen, and E. Terasawa Rapid Action of Estrogens on Intracellular Calcium Oscillations in Primate Luteinizing Hormone-Releasing Hormone-1 Neurons Endocrinology, March 1, 2008; 149(3): 1155 - 1162. [Abstract] [Full Text] [PDF]
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I. Nishimura, K. Ui-Tei, K. Saigo, H. Ishii, Y. Sakuma, and M. Kato 17{beta}-Estradiol at Physiological Concentrations Augments Ca2+-Activated K+ Currents via Estrogen Receptor {beta} in the Gonadotropin-Releasing Hormone Neuronal Cell Line GT1-7 Endocrinology, February 1, 2008; 149(2): 774 - 782. [Abstract] [Full Text] [PDF]
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A. Morales, M. Gonzalez, R. Marin, M. Diaz, and R. Alonso Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells J. Endocrinol., July 1, 2007; 194(1): 193 - 200. [Abstract] [Full Text] [PDF]
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J. Lindzey, F. L Jayes, M. M Yates, J. F Couse, and K. S Korach The bi-modal effects of estradiol on gonadotropin synthesis and secretion in female mice are dependent on estrogen receptor-{alpha}. J. Endocrinol., October 1, 2006; 191(1): 309 - 317. [Abstract] [Full Text] [PDF]
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D. Titolo, F. Cai, and D. D. Belsham Coordinate Regulation of Neuropeptide Y and Agouti-Related Peptide Gene Expression by Estrogen Depends on the Ratio of Estrogen Receptor (ER) {alpha} to ER{beta} in Clonal Hypothalamic Neurons Mol. Endocrinol., September 1, 2006; 20(9): 2080 - 2092. [Abstract] [Full Text] [PDF]
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J. P. Cabilla, M. d. C. Diaz, L. I. Machiavelli, A. H. Poliandri, F. A. Quinteros, M. Lasaga, and B. H. Duvilanski 17{beta}-Estradiol Modifies Nitric Oxide-Sensitive Guanylyl Cyclase Expression and Down-Regulates Its Activity in Rat Anterior Pituitary Gland Endocrinology, September 1, 2006; 147(9): 4311 - 4318. [Abstract] [Full Text] [PDF]
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T. R. Pak, W. C. J. Chung, J. L. Roberts, and R. J. Handa Ligand-Independent Effects of Estrogen Receptor {beta} on Mouse Gonadotropin-Releasing Hormone Promoter Activity Endocrinology, April 1, 2006; 147(4): 1924 - 1931. [Abstract] [Full Text] [PDF]
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H. Abe and E. Terasawa Firing Pattern and Rapid Modulation of Activity by Estrogen in Primate Luteinizing Hormone Releasing Hormone-1 Neurons Endocrinology, October 1, 2005; 146(10): 4312 - 4320. [Abstract] [Full Text] [PDF]
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 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, A. Bae, R. S. Swerdloff, A. Iranmanesh, and C. Wang Experimentally Induced Androgen Depletion Accentuates Ethnicity-Related Contrasts in Luteinizing Hormone Secretion in Asian and Caucasian Men J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1632 - 1638. [Abstract] [Full Text] [PDF]
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A. van de Stolpe, A. J. Slycke, M. O. Reinders, A. W. M. Zomer, S. Goodenough, C. Behl, A. F. Seasholtz, and P. T. van der Saag Estrogen Receptor (ER)-Mediated Transcriptional Regulation of the Human Corticotropin-Releasing Hormone-Binding Protein Promoter: Differential Effects of ER{alpha} and ER{beta} Mol. Endocrinol., December 1, 2004; 18(12): 2908 - 2923. [Abstract] [Full Text] [PDF]
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 D. M. Selva, O. M. Tirado, N. Toran, C. A. Suarez-Quian, J. Reventos, and F. Munell Estrogen Receptor {beta} Expression and Apoptosis of Spermatocytes of Mice Overexpressing a Rat Androgen-Binding Protein Transgene Biol Reprod, November 1, 2004; 71(5): 1461 - 1468. [Abstract] [Full Text] [PDF]
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J. S. Jorgensen, C. C. Quirk, and J. H. Nilson Multiple and Overlapping Combinatorial Codes Orchestrate Hormonal Responsiveness and Dictate Cell-Specific Expression of the Genes Encoding Luteinizing Hormone Endocr. Rev., August 1, 2004; 25(4): 521 - 542. [Abstract] [Full Text] [PDF]
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C. E. Navarro, S. A. Saeed, C. Murdock, A. J. Martinez-Fuentes, K. K. Arora, L. Z. Krsmanovic, and K. J. Catt Erratum Mol. Endocrinol., December 1, 2003; 17(12): A - 2658. [Abstract] [Full Text] [PDF]
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S. L. Petersen, E. N. Ottem, and C. D. Carpenter Direct and Indirect Regulation of Gonadotropin-Releasing Hormone Neurons by Estradiol Biol Reprod, December 1, 2003; 69(6): 1771 - 1778. [Abstract] [Full Text] [PDF]
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C. E. Navarro, S. Abdul Saeed, C. Murdock, A. J. Martinez-Fuentes, K. K. Arora, L. Z. Krsmanovic, and K. J. Catt Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling and Pulsatile Neurosecretion by Gi-coupled Plasma Membrane Estrogen Receptors in Immortalized Gonadotropin-Releasing Hormone Neurons Mol. Endocrinol., September 1, 2003; 17(9): 1792 - 1804. [Abstract] [Full Text] [PDF]
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N. R. Thanky, R. Slater, and A. E. Herbison Sex Differences in Estrogen-Dependent Transcription of Gonadotropin-Releasing Hormone (GnRH) Gene Revealed in GnRH Transgenic Mice Endocrinology, August 1, 2003; 144(8): 3351 - 3358. [Abstract] [Full Text] [PDF]
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J. G. Ramos, J. Varayoud, L. Kass, H. Rodriguez, L. Costabel, M. Munoz-de-Toro, and E. H. Luque Bisphenol A Induces Both Transient and Permanent Histofunctional Alterations of the Hypothalamic-Pituitary-Gonadal Axis in Prenatally Exposed Male Rats Endocrinology, July 1, 2003; 144(7): 3206 - 3215. [Abstract] [Full Text] [PDF]
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S. Khosravi and P. C. K. Leung Differential Regulation of Gonadotropin-Releasing Hormone (GnRH)I and GnRHII Messenger Ribonucleic Acid by Gonadal Steroids in Human Granulosa Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 663 - 672. [Abstract] [Full Text] [PDF]
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R. Vazquez-Martinez, G. M. Leclerc, M. E. Wierman, and F. R. Boockfor Episodic Activation of the Rat GnRH Promoter: Role of the Homeoprotein Oct-1 Mol. Endocrinol., September 1, 2002; 16(9): 2093 - 2100. [Abstract] [Full Text] [PDF]
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N. Sharifi, A. E. Reuss, and S. Wray Prenatal LHRH Neurons in Nasal Explant Cultures Express Estrogen Receptor {beta} Transcript Endocrinology, July 1, 2002; 143(7): 2503 - 2507. [Abstract] [Full Text] [PDF]
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 S. Lyou, E. Hirano, K. Tujioka, Y. Mawatari, K. Hayase, S. Okuyama, and H. Yokogoshi Dietary Genistein Affects Brain Protein Synthesis Rates in Ovariectomized Female Rats J. Nutr., July 1, 2002; 132(7): 2055 - 2058. [Abstract] [Full Text] [PDF]
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S. Gill, J. L. Sharpless, K. Rado, and J. E. Hall Evidence That GnRH Decreases with Gonadal Steroid Feedback but Increases with Age in Postmenopausal Women J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2290 - 2296. [Abstract] [Full Text] [PDF]
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 M. Potier, M. Karl, F. Zheng, S. J. Elliot, G. E. Striker, and L. J. Striker Estrogen-Related Abnormalities in Glomerulosclerosis-Prone Mice : Reduced Mesangial Cell Estrogen Receptor Expression and Prosclerotic Response to Estrogens Am. J. Pathol., May 1, 2002; 160(5): 1877 - 1885. [Abstract] [Full Text] [PDF]
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 K. M. Waters, S. Safe, and K. W. Gaido Differential Gene Expression in Response to Methoxychlor and Estradiol through ER{alpha}, ER{beta}, and AR in Reproductive Tissues of Female Mice Toxicol. Sci., September 1, 2001; 63(1): 47 - 56. [Abstract] [Full Text] [PDF]
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T. R. Pak, G. R. Lynch, and P.-S. Tsai Testosterone and Estrogen Act via Different Pathways to Inhibit Puberty in the Male Siberian Hamster (Phodopus sungorus) Endocrinology, August 1, 2001; 142(8): 3309 - 3316. [Abstract] [Full Text] [PDF]
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 M. POTIER, S. J. ELLIOT, I. TACK, O. LENZ, G. E. STRIKER, L. J. STRIKER, and M. KARL Expression and Regulation of Estrogen Receptors in Mesangial Cells: Influence on Matrix Metalloproteinase-9 J. Am. Soc. Nephrol., February 1, 2001; 12(2): 241 - 251. [Abstract] [Full Text]
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 J. S. Krumenacker, S. M. Hyder, and F. Murad Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus PNAS, January 16, 2001; 98(2): 717 - 722. [Abstract] [Full Text] [PDF]
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K. Hayase, M. Tanaka, K. Tujioka, E. Hirano, O. Habuchi, and H. Yokogoshi 17-{beta}-Estradiol Affects Brain Protein Synthesis Rate in Ovariectomized Female Rats J. Nutr., January 1, 2001; 131(1): 123 - 126. [Abstract] [Full Text]
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F. J. Hayes, S. B. Seminara, S. DeCruz, P. A. Boepple, and W. F. Crowley Jr. Aromatase Inhibition in the Human Male Reveals a Hypothalamic Site of Estrogen Feedback J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3027 - 3035. [Abstract] [Full Text]
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