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Protein and Estrogen Responsiveness1
Department of Internal Medicine, Division of Endocrinology and Metabolism, and Department of Pathology (M.H.S.), University of Virginia, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 800578, Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville, Virginia 22908. E-mail: mas3x{at}virginia.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ERß, and the
pituitary-specific ER isoform, truncated estrogen receptor product-1
(TERP-1). In cycling female rat pituitaries, ERß messenger RNA (mRNA)
levels fell 40% on the morning of proestrus and were suppressed by E
or dihydrotestosterone in ovariectomized females. In lactotrope and
gonadotrope cell lines (GH3, RC4B, LßT2), progesterone
(P) or P plus E also suppressed ERß. TERP-1 mRNA increased 3-fold at
proestrus and in response to E treatment in vivo and in
cell lines. ER mRNA levels were not regulated significantly by any
treatment in vivo or in cell lines. However, E
suppressed ER protein levels in vivo and in cell
lines, and reduction of ER protein levels by E or the antiestrogen
ICI182,780 reduced E-stimulated transcriptional activation of the PRL
promoter in GH3 cells. TERP-1 and ERß protein levels were
low to undetectable in cell lines, but E stimulated TERP-1. Because E
treatment decreases ERß mRNA and ER protein and increases levels
of TERP-1 (which can suppress ER/ß activity), the dynamic
steroid-induced changes in ER expression in the rat pituitary during
the midcycle gondaotropin/PRL surge may provide a means for ovarian
steroids to limit positive feedback. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen acts to modulate gene expression through estrogen receptors
(ERs), which belong to a superfamily of nuclear transcription factors
(4). These proteins have a conserved domain structure with a centrally
located DNA-binding domain (DBD), an N-terminal ligand-independent
trans-activating function (AF1), and a C-terminal
ligand-binding domain (LBD) with a ligand-dependent
trans-activating function (AF2). Ligand-bound ERs form
dimers that act on specific estrogen response elements in the promoter
regions of E-regulated genes. At least three major ER isoforms are
expressed in the rat pituitary including ER, ERß, and truncated
estrogen receptor product-1 (TERP-1) (5, 6). ER and ERß share 95%
homology in the DBD, approximately 50% homology in the LBD, and little
homology in the N-terminus (7). TERP-1 contains a unique
5'-untranslated sequence fused to exons 5–8 of the ER (5).
Translation of TERP-1 messenger RNA (mRNA) leads to a protein with most
of the LBD of ER, but no N-terminus or DBD (5, 8). In transcription
assays, the relative expression of these receptors can have profound
effects on estrogen-induced trans-activation. Specifically,
ER has greater trans-activating ability than ERß (9, 10), and the binding affinity and activity of certain phytoestrogens
and synthetic estrogen ligands differ for ER and ERß (11).
Although TERP-1 has no independent activity, it can stimulate ER
activity at low ratios and inhibit the activity of ER and ERß at
ratios of 1:1 or greater (12, 13). Thus, the overall response of
tissues and regulated genes to E is in part dependent on the relative
expression of ER isoforms.
The tissue-specific expression of ER mRNAs has been examined
extensively in several species. Many estrogen-responsive tissues
express both ER and ERß, although urogenital structures and
lung express significantly more ERß (14). Previous studies have shown
that all three ER mRNAs are present in rat pituitaries, with ER
sequences present at much higher levels than ERß (6, 15, 16). TERP-1
expression is limited to the pituitary (5, 17, 18). A recent in
situ hybridization study has demonstrated the presence of ER in
several pituitary cell types, including gonadotropes, lactotropes, and
corticotropes (6). ERß mRNA also appears to be widely distributed in
the rodent pituitary (6, 15, 16, 19). Interestingly, ERß is the major
pituitary ER expressed in prepubertal female rats, and mRNA levels in
females are almost twice those seen in males at this time (16).
Similarly, under physiological conditions TERP-1 mRNA is expressed only
in female rat pituitaries (8, 18).
Little is known about the promoter and regulatory regions of ERs,
although several alternate first exons and promoters exist for ER
and ERß (20, 21). TERP-1 mRNA is transcribed from a unique
transcriptional start site, allowing divergent and cell-specific
regulation of ER forms. In a recent report, ER and ERß mRNAs in
adult rat pituitaries were not altered by E (6); however, we and others
have found modulation of ER and TERP-1 mRNA through the estrous
cycle (8, 18). Regulation of ERß expression across the estrous cycle
has not been described.
In the present study we examined the regulation of ER isoforms in the
rat pituitary during the estrous cycle and in response to different
steroids and hypothalamic peptides and compared these results with the
regulation of ER isoforms in lactotrope and gonadotrope clonal cell
lines. Our results demonstrate that there is a significant regulation
of ER isoforms in pituitary cells in vivo and in
vitro and that the regulation of ERs by steroids and hypothalamic
peptides is cell type specific. Furthermore, E specifically suppresses
ER protein levels, and this suppression diminishes subsequent E
responsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (DHT; 20 µg/100 g BW). Ribonuclease (RNase)
protection data for ER and TERP-1 were previously reported for these
animals (8). An additional four ovariectomized female rats were used
for assessment of ER and TERP protein levels after treatment with
oil vehicle (n = 2) or E (n = 2) as described above. For
antagonist experiments ovariectomized female rats were injected sc for
5 days with oil vehicle or 17ß-estradiol (30 µg) at 0930 h. On
days 3–5, one E-treated group also received 250 µg of the dopamine
agonist bromocriptine (Sigma, St. Louis, MO)
in PBS, and one group received 50 µg of the GnRH antagonist antide
(Sigma) in 20% propylene glycol at 0930 h. On day 5,
animals were killed by decapitation at 1430 h. Pituitaries were
removed for isolation of RNA, and trunk blood was collected for assay
of LH and PRL to confirm the effectiveness of treatments. For in
situ hybridization experiments castrate male and female rats were
injected for 3 days with oil vehicle or E (30 µg). In all cases,
animals were anesthetized with halothane and killed by
decapitation.
In situ hybridization
Pituitaries were fixed in 4% paraformaldehyde and embedded in
paraffin. Five-micron tissue sections were mounted on
3-aminopropyl-thriethoxysilane-coated glass microscope slides.
Alternate sections were subjected to in situ hybridization
with tritium-labeled antisense riboprobes for rat LHß, ß-actin, the
N-terminus of rat ER, the C-terminus of rat ER, and PRL.
Additional sections were processed with sense probes or were treated
with RNase. Riboprobes for LHß (325 bp) and PRL (467 bp) were
prepared from full-length coding sequence in pGem7 vector
(Promega Corp., Madison, WI) and PCR2.1 vector
(Invitrogen, San Diego, CA), respectively. The actin probe
was prepared from a full-length clone of chicken ß-actin (1.8 kb).
Riboprobes for rat ER were prepared by transcription from cloned
fragments of ERs in the pGem7 vector. Probes corresponding to the
N-terminus untranslated region to exon 4 (710 bp) and the C-terminus
from exons 5–8 (770 bp) were used. Probes were matched with respect to
coding sequences and C plus U content. The N-terminal probe recognizes
ER, and the C-terminal probe recognizes both ER and TERP. In
addition, a full-length ERß (11) riboprobe was prepared from coding
sequence in pGem3Z. Probes were prepared using the appropriate
polymerase in the presence of a mixture of 50
µM deoxy-CTP and 50 µM
deoxy-UTP with a specific activity of approximately 3.4 x
107 cpm/µg. Probes (except LHß) were
hydrolyzed to a desired length of 300 bp and hybridized at 55 C. Slides
were washed in 0.1 x saline-sodium citrate at 65 C and coated
with Kodak NTB-2 autoradiography emulsion (Eastman Kodak Co., Rochester, NY). Slides were counterstained with
hematoxylin and eosin. Autoradiographic signals were photographed under
darkfield illumination, and histology was reviewed with brightfield
illumination. Hybridization signal was quantified as previously
described (22). Briefly, 8-bit gray scale digital images were acquired
with a Gould IP850 Image Processing Workstation (Gould Electronics,
Inc., Fremont, CA) with spatial resolution of 512 x 512 pixels.
Data were analyzed on a Sun-3 workstation (Sun Microsystems, Inc.,
Mountain View, CA) the using the ANALYZE software package (Biodynamics
Research Unit, Mayo Foundation, Rochester, MN). A threshold for
positive cells was selected at a half-maximum difference between
background grain intensity and grain intensities over positive cells.
The threshold was used to outline an area of confluent grains above
each cell, and the grain intensity of this area was quantified.
Cells lines, chemicals, and treatments
Mouse- and rat-derived pituitary cell lines were used. Rat
somatomammotrope GH3 cells and rat lactotrope MMQ
and PR1 cells were maintained in DMEM/10% newborn calf serum (NCS;
Life Technologies, Inc., Gaithersburg, MD). Mouse T3
pregonadotrope and LßT2 differentiated gonadotrope cells were
maintained in DMEM/10% FBS (Life Technologies, Inc.). Rat
lactogonadotrope RC4B cells were maintained in 50% DMEM/50% MEM with
10% FBS, 0.02% BSA, 400 pM epidermal growth factor, and
15 mM HEPES (pH 7.6). All media contained 100 U/ml
penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). Before treatments cells were split into culture flasks
(75 mm2; Corning, Inc., Corning, NY)
or 60-mm four-well culture plates (ICN Biomedicals, Inc.,
Costa Mesa, CA) for 1 day in maintenance medium. The following day,
medium was removed, cells were washed twice with PBS, and medium was
replaced with phenol red-free DMEM/5% charcoal-stripped NCS. Cells
were maintained in this medium for 4 days during treatments. Cells were
left untreated (NT), treated for 1 or 4 days with 10 nM E
(Sigma), treated for 4 days with 10 nM DHT
(Sigma), treated for 4 days with 0.1 µM P
(Sigma), or treated for 4 days with E and P. For pituitary
adenylate cyclase activating peptide treatment, 100
nM PACAP (Sigma) was added for the final
18 h of treatment; for GnRH treatment, 50 nM GnRH
(Sigma) was added for the final 6 h of incubation.
After treatments, cells were washed twice with PBS and collected as
described below. Two separate treatment groups were prepared for each
cell line. For RT-PCR analysis, each treatment group was amplified two
or four times from separate RT reactions. For immunoblotting, three or
four separate treatment groups were prepared and analyzed.
Transient transfections
GH3 cells were transiently transfected
with 2 µg of a reporter construct containing 2.5 kb of the
estrogen-responsive rat PRL promoter fused to a luciferase
reporter gene (gift from Dr. Richard Day), as previously described
(12). Cells were plated in DMEM/5% charcoal-stripped NCS with or
without 10 nM E. Untreated parallel wells were subsequently
treated 18 h later with the antiestrogen ICI 182,780 (10
nM). Cells were transfected 6 h later. After 2 h
in diethylaminoethyl-dextran transfection medium,
GH3 cells were shocked for 2 min with 10%
dimethylsulfoxide and subsequently treated with or without E for 22 h.
Parallel wells were collected for protein determination by
immunoblotting.
Semiquantitative RT-PCR
RNA was extracted from cells by lysis in guanidinium thiocyanate
as previously described (8). Conditions for semiquantitative RT-PCR
were determined separately for each pituitary cell line and rat
pituitary samples because the relative abundance of ER message varied
among tissues and pituitary cell lines. Total RNA was reverse
transcribed in a 20-µl mixture consisting of 5 mM
MgCl2 (Perkin-Elmer Corp., Palo
Alto, CA), PCR buffer II, 2 mM deoxyribonucleotides, 1 U
RNase inhibitor, 2.5 µM random hexamers, and 2.5 U murine
leukemia virus reverse transcriptase. RT reactions were incubated 10
min at room temperature, 15 min at 42 C, and 5 min at 99 C and cooled
to 4 C for 5 min in a Perkin-Elmer Corp. Thermocylcer 480.
For PCR reactions, MgCl2 was adjusted to 2
mM, and buffer, water, 0.2 mM primer
oligonucleotides (Operon Technologies), and 2.5 U/100 µl
Taq DNA polymerase (Sigma) were added to a
final volume of 16–100 µl. PCR was performed in a
Stratagene Robocycler Gradient 40. General PCR conditions
consisted of a single cycle of 3 min at 95 C, 3 min at annealing
temperature, and 3 min at 72 C. Additional cycles were performed with
45-sec steps and a final 10-min extension step at 72 C. Optimization
was performed separately for each set of primers. Annealing temperature
(56–66 C) for each primer set was determined at 35 cycles with 0.5–8
µg input RNA. Optimal cycle number was determined over a range of
15–40 cycles. For cell lines, 640 ng input RNA fell in the linear
range for all primer sets. For pituitaries 160 ng input RNA were used.
Primer sequences, the expected product sizes, and PCR conditions are
shown in Table 2
. Primers were chosen to recognize both rat and mouse
sequences and to cross intron boundaries. ER was amplified over a
range of 20 (T3) to 30 cycles (LßT2), ERß was amplified over a
range of 32 (LßT2, GH3) to 37 cycles (RC4B), and TERP was amplified
over a range of 33 (rat pituitary) to 36 (T3) or 37 cycles (all
others). Both ER and ERß primer sets were directed against
N-terminal regions, and thus detect both the full-length receptors and
certain C-terminal variants of ER (exon 5/6 deletions) and ERß.
Previous results (5, 8) suggest that ER variants in the rat
pituitary are not highly regulated and represent only a small
proportion of the ER mRNA population. In vivo treatment
samples were also assessed for regulation of the ERß2 LBD 54-bp
insertion variant (23) using PCR primers 5' and 3' to the region of the
insert. PCR amplification over 37 cycles resulted in products of 371 bp
for ERß1 and 425 bp for ERß2. A unique EAR1 restriction site in the
insert was used to verify the identity of ERß2. RPL19 ribosomal
protein mRNA primers were included in each PCR reaction as an internal
control (24). The linear range for RPL19 mRNA was also determined
separately for each cell line (data not shown). For ER and ERß,
RPL19 was amplified over the same cycle number as the ER of interest.
However, in the case of TERP-1, RPL19 primers were added to the
reaction mixture after four to seven cycles to allow sufficient initial
synthesis of TERP-1 templates. After PCR, 16 µl of each reaction were
separated on 1.5% agarose gels containing ethidium bromide (0.7
µg/ml). Gels were photographed and evaluated by fluoroimaging with a
Molecular Dynamics, Inc. (Menlo Park, CA), Fluoroimager
515. Data were analyzed with ImageQuant software (Molecular Dynamics, Inc.).
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and TERP-1 protein expression in cell lines was determined
from 50 µg total protein and separated on 12% polyacrylamide-SDS
gels by immunoblot analysis using a rabbit polyclonal antibody as
previously described (12). The antibody (C1355) was generated against
C-terminal amino acids 586–600 of the rat ER and also detects mouse
ER (8). Western analysis was performed with enhanced
chemiluminescence (Amersham Pharmacia Biotech, Arlington
Heights, IL) using the C1355 primary antibody at 1:2,500 for 1 h,
followed by a 1-h incubation with a horseradish peroxidase-conjugated
donkey antirabbit IgG secondary antibody (Amersham Pharmacia Biotech) at 1:800. For rat pituitaries, 75 µg total protein
were used, and primary antibody was used at 1:5,000. Protein loading
was normalized to ß-actin using a monoclonal primary antibody at
1:5,000 (Sigma) and a horseradish peroxidase-conjugated
goat antimouse IgG secondary antibody at 1:40,000 (The Jackson Laboratory, Bar Harbor, ME). In some cases, in vitro
translated proteins or proteins from transfected COS cells were
included on blots as markers and positive controls for experimental
conditions. Densitometry was performed with a Molecular Dynamics, Inc., Personal Densitometer SI and analyzed with ImageQuant
software (Molecular Dynamics, Inc.).
Statistical analysis
Data for each ER and each cell line or animal experiment were
analyzed separately. After one-way ANOVA, a priori pairwise
comparisons were made between treatment groups and controls using
t tests. For in vivo estrous cycle experiments,
all days were compared with metestrus. For the remainder of the
in vivo experiments comparisons were made to the oil-treated
controls. For cell lines, comparisons were made between treatments and
the untreated condition. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TERP, and ERß by E in pituitary slices.
Because TERP-1 differs from ER only in the first 31 bp, expression
was determined using both an N-terminal (detects ER only) and a
C-terminal riboprobe to ER (detects ER and TERP-1). Other TERP
mRNA species have been detected at lower abundance than TERP-1 and may
contribute to the C-terminal signal; however, only TERP-1 appears to be
expressed at significant levels and translated into protein (5, 8, 17, 25). Therefore, differences in regulation between the two signals can
be accounted for by TERP-1 expression. In ovariectomized female rats, E
treatment decreased LHß expression and increased PRL expression as
expected (Fig. 1, A–D, and Table 1). Treatment of castrate males showed
similar results in three experiments (data not shown). ER expression
determined with the N-terminal riboprobe was more diffuse than hormone
mRNAs, but overlapped regions containing gonadotropes and lactotropes
(Fig. 1E and Table 1). E treatment had no significant effect on ER
expression throughout the pituitary, although there was a slight
decrease in hybridization signal (Fig. 1F and Table 1). However, E
treatment led to a 2.9-fold increase in C-terminal message expression
(Fig. 1H and Table 1). Increased signal intensity was observed
throughout the slices, and dramatic alteration in specific cell
populations was not observed. These data support the observation that
TERP is up-regulated by E even when full-length ER levels are
unchanged. ERß mRNA was expressed at much lower levels than ER and
appeared to be more diffuse (data not shown). ERß levels were close
to background and were not significantly modulated by E.
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shows the relative expression of
ER, TERP, and ERß from 15–40 cycles of PCR amplification in rat
pituitary and cell lines. Figure 2B shows the expression curves for
each mRNA isoform with 40 cycles normalized to 100%. In subsequent
studies with rat pituitaries, ER was amplified over 29 cycles, ERß
over 36 cycles, and TERP over 33 cycles (see Table 2
and Fig. 2). ER mRNA, representing
full-length ER and ER exon 5/6 deletions, did not change
significantly across the estrous cycle (Fig. 3). The exon 5/6 deletion is present at
low levels (10% of full length) and is not regulated by steroids (8).
In contrast, TERP-1 mRNA expression rose 3.2-fold over metestrus levels
by the morning of proestrus (Fig. 3), and ERß levels fell 40% on the
morning of proestrus compared with levels on metestrus, coincident with
the peak of TERP-1 expression (Fig. 3). These ERß values represent
both ERß1 and ERß2, a variant containing a 54-bp insertion in the
LBD.
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mRNA is not highly
regulated by E or P, but TERP-1 mRNA is induced dramatically by E (Fig. 4 and Ref. 8). In addition, we found that
DHT significantly increased TERP-1 mRNA levels in female rats (Fig. 4A). P treatment had no effect on TERP-1 expression and did not alter
the effect of E in females (Fig. 4, A and C) or males (data not shown).
Treatments that increased TERP-1 mRNA in females (E, E plus P, and DHT)
also significantly decreased ERß mRNA expression, but had no effect
on ER (Fig. 4A). Similar trends were observed in E-treated and E-
plus P-treated males (data not shown). ERß values represent both
ERß1 and ERß2, a variant containing a 54-bp insertion in the LBD.
An additional primer set was used to coamplify ERß1 and ERß2. Both
variants were regulated in a similar manner, and ERß2 was expressed
at slightly higher levels than ERß1 (Fig. 4B). Immunoblots and
immunohistochemical studies with available ERß antibodies indicate
that ERß protein levels are very low, and regulation could not be
assessed (data not shown).
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mRNA levels were unaffected by any treatment, but E
suppressed ERß mRNA and stimulated TERP-1 mRNA (Fig. 4C).
Neither antide nor bromocriptine altered these responses
to E.
Physiological pituitary responses to E are ultimately dependent on the
expression levels of ER protein. Because mRNA levels, translational
efficiency, and protein turnover influence ER protein levels, we
examined whether changes seen at the mRNA level were also reflected at
the level of protein expression. Although ER mRNA levels were not
altered by steroid treatments in ovariectomized female rats,
immunoblotting revealed that ER protein levels were reduced 3-fold
in E-treated rats (Fig. 5). In contrast,
TERP protein was essentially undetectable in oil-treated animals and
was increased nearly 6-fold by E treatment (Fig. 5). Thus, the ratio of
TERP to ER in oil-treated animals was 0.3 and rose to 1.2 with
E.
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shows the relative expression of
ER, ERß, and TERP mRNAs by PCR amplification in a rat
somatolactotrope cell line (GH3), rat lactotrope
cell lines (PR1 and MMQ), a rat lactogonadotrope cell line (RC4B), a
mouse gonadotrope precursor cell line (T3), and a mouse
differentiated gonadotrope cell line (LßT2). For comparison, ER
expression in an E-treated male rat is shown (120 ng input RNA/lane
compared with 640 ng for cell lines). All cell lines expressed high
levels of ER, with detectable expression after 20 cycles of PCR.
ERß was relatively more abundant in cells with lactotrope
characteristics (GH3, PR1, and MMQ), although it
was clearly present in all cell types. An additional smaller
amplification product was detected for ERß in T3 cells, but
Southern analysis with a nested probe did not hybridize to this product
(data not shown). Therefore, we believe it is a nonspecific
amplification. TERP-1 was most highly expressed in MMQ and RC4B cells.
MMQ and PR1 cells also expressed high levels of TERP-2 mRNA, a larger
TERP splice variant that contains additional intronic sequence between
the 31-bp TERP-specific sequence and exon 5 of ER (5). In general,
cells with lactotrope characteristics expressed the highest levels of
TERP, consistent with the in vivo expression of TERP in
normal lactotropes (18). TERP mRNA was not expressed in two other
pituitary cell lines, AtT20 corticotropes and TSH thyrotrope
precursors (not shown).
Both steroid (E, DHT, P, and P plus E) and peptide (PACAP and
GnRH) treatments were employed to examine the cell type-specific
regulation of ER isoforms in GH3, RC4B, T3,
and LßT2 cells. No significant changes in ER expression were observed
in T3 cells with any treatment, and ER mRNA was not significantly
altered by any treatment in cells with lactotrope characteristics
(GH3 and RC4B; Fig. 6, A and B). However, P in combination
with E increased ER mRNA levels in the differentiated gonadotrope
LßT2 cell line, and the effect of P alone did not reach significance
(P < 0.06; Fig. 6C). ERß mRNA levels were suppressed
by P alone (LßT2) or P plus E (GH3; Fig. 6), but this effect was not
significant in RC4B cells. TERP-1 mRNA was consistently up-regulated by
1 or 4 days of E in pituitary cell lines, in agreement with previous
results noted in vivo (Fig. 6). In the lactotropic cell
lines (GH3 and RC4B) DHT also increased TERP-1
expression, in agreement with our in vivo data (Fig. 6, A
and B). No such increase was seen in LßT2 cells (Fig. 6C), even
though these cells possess functional androgen receptors (our
unpublished observations). Conversely, in cell lines with gonadotropic
characteristics (RC4B and LßT2), P increased TERP-1 expression (Fig. 6, B and C). Expression of TERP-1 was unaffected by GnRH treatment in
cell lines that express GnRH receptors (LßT2 and RC4B; data not
shown), and GnRH did not affect E-induced TERP expression. In contrast,
PACAP stimulated TERP-1 mRNA in GH3 and LßT2
cells (Fig. 6, A and C).
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protein was readily detected in all cell types, with
differential expression among cells. Cells with the highest levels of
ER mRNA (T3 and GH3) had the highest levels of ER
protein. Estrogen treatment decreased ER protein in all cell types
between 25–60%, and this decrease was also observed in the presence
of E and P (Fig. 7A and Table 3), even though no changes were observed
in mRNA expression. These results were confirmed in duplicate
immunoblots from two or three separate treatment groups. TERP-1 protein
levels were very low relative to ER and were detectable in only some
experiments in T3, RC4B, and LßT2 cells. However, E stimulated
TERP-1 protein when it was detected (Fig. 7B). DHT also increased
TERP-1 protein expression in RC4B, LßT2, and T3 cells. In
agreement with mRNA expression data in LßT2 cells, P alone increased
TERP-1 protein, but PACAP had no significant effect. Thus, in general,
stimulation of TERP-1 protein occurred in parallel with mRNA; however,
ER protein, but not mRNA, was significantly suppressed by E
treatment. ERß protein could not be reliably detected in cell lines
using two commercially available antibodies; however, ERß could be
detected in transfected COS-1 cells and rat ovary. Thus, ERß protein
levels in pituitary cell lines are likely to be very low.
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protein on estrogen- mediated
transcription protein levels
would reduce E responsiveness in pituitary cells. Pretreatment of
GH3 cells with 10 nM E markedly
diminished E-induced transcription of a PRL promoter-containing
reporter gene (Fig. 8). In addition to
competing with E for ER binding, the antiestrogen ICI182,780 increases
ER turnover and reduces ER levels in the nucleus (27). Pretreatment of
GH3 cells with ICI182,780, reduced E
responsiveness (Fig. 8). Both E and ICI182,780 pretreatments markedly
reduced ER protein levels (Fig. 8). After an additional 24 h of
treatment with E for determination of reporter gene activity, ER
levels were all similarly reduced (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ERß, and TERP-1.
Both ER and ERß have been reported to be transcribed from several
alternate first exons and multiple promoters (20, 21, 28, 29). TERP-1
is transcribed from a promoter distinct from those for ER (5, 25).
Thus, these three isoforms can be differentially regulated and may
result in different transcriptional capacities. In model systems, ER
stimulates E-regulated genes better than ERß (10, 30), and TERP-1
modifies the activity of full-length ERs (12). Previous studies (5, 6, 8, 17, 18) showed that E regulates ER isoforms in the rat pituitary.
The present study demonstrates that pituitary TERP-1 and ERß mRNA
levels can be physiologically regulated in vivo as well as
in cell lines by steroid hormones and other peptides. Similarly, ER
and TERP-1 protein levels are also modulated. These results have
implications for steroid feedback during the estrous cycle and may
provide one means for termination of the proestrous LH and PRL surge by
reducing the transcriptional capacity of ER.
To begin to elucidate the cell-specific expression and regulation of
ERs in the pituitary, we used two in vivo treatments and
examined rodent pituitary cell lines of various lineages. Previous
studies have shown that ER is expressed primarily in lactotropes and
gonadotropes. In the normal human pituitary, ER mRNA is expressed in
50% of lactotropes, 70–83% of gonadotropes (31), and fewer than 5%
of somatotropes, thyrotropes, or corticotropes (31). In the adult
female rat, ER mRNA is expressed in 45% of lactotropes and 25% of
gonadotropes (6). In humans, ERß is present in pituitary adenomas of
every cell type and is the only ER form expressed in tumors that
express GH exclusively (32, 33). ERß expression in rodent pituitaries
suggests that levels are extremely low. Although we detected ERß mRNA
by RT-PCR from whole pituitary, we did not detect significant levels of
ERß mRNA using tritiated riboprobes. Similarly, Couse et
al. (19) did not detect ERß mRNA in mouse pituitaries using
RNase protection, although ERß has been detected in the intermediate
lobe of the adult rat pituitary using 35S-labeled
riboprobes (15). However, Wilson et al. (16) recently
demonstrated that prepubertal female rats have a relative abundance of
ERß in the pituitary, and 15% of lactotropes and 85% of
FSH-positive gonadotropes express ERß mRNA at low levels. Similarly,
Mitchner and colleagues (6) concluded that 25% of lactotropes and 15%
of LH-expressing gonadotropes in the adult female pituitary contain
ERß mRNA. Using RT-PCR we detected ERß mRNA in all pituitary cell
lines tested, but the pattern of expression differed from that of
ER. Cells with more lactotropic characteristics
(GH3, PR1, and MMQ) expressed significantly more
ERß than cells with gonadotrope characteristics (T3 and LßT2).
ERß protein was not detected in any of the cell lines, possibly due
to both low expression levels and the relative insensitivity of the
ERß antibodies. TERP-1 mRNA has been reported in enriched rat
lactotrope populations, but is absent in enriched gonadotrope
populations using Northern blot analysis (18). The present study
suggests that TERP-1 may be present at some level in both lactotropes
and gonadotropes, but lactotrope cell lines express more TERP-1
mRNA.
ER mRNA levels did not change across the estrous cycle or in
response to most treatments in vivo or in cell lines, in
agreement with previous studies showing only moderate to no regulation
in vivo and in GH3 cells (8, 18, 34).
Previous data from our laboratory demonstrated that total ER (ER
and TERP-1) mRNA on Northern blots was stimulated by E treatment (5),
and subsequent studies demonstrated that TERP-1 mRNA was differentially
stimulated by E (5, 8). Although we observed a 2- to 3-fold E-induced
stimulation of ER by RNase protection, our RT-PCR product
representing ER and the ER exon 5/6 deletion variant mRNA was not
changed. We believe that in part this is due to a larger dynamic range
for the RNase protection. Thus, the change in TERP-1 mRNA expression
observed in the present study is also smaller than that observed
previously (5, 8). This is probably due to better detection of low
levels of expression with RT-PCR. Interestingly, other investigators
using RT-PCR found a similar lack of effect of E on ER (34).
Immunoblotting demonstrated that full-length ER protein levels could
be highly regulated in vivo in the pituitary, even in the
face of relatively steady state mRNA levels. Other studies have shown
small reductions in E binding in the pituitary around proestrus,
suggesting that total ER protein levels are reduced (35, 36, 37). The
decrease in ER protein levels provides one mechanism by which
E-induced positive feedback may be limited, and such
posttranscriptional changes in ER may be an important mechanism for
ER regulation in both the pituitary and other tissues (38). In the
present study ER protein was readily suppressed by E in all
pituitary cell lines tested, including PR1 cells (data not shown).
Dissociation of mRNA and protein expression may provide an important
mechanism for cell-specific actions of E. Recent findings by Alarid
et al. (38) show that in PR1 pituitary cells the reduction
of ER in response to E occurs via proteosomal degradation. A similar
dissociation of mRNA and protein expression has been demonstrated for
the androgen receptor (39) and thyroid hormone receptor (40, 41).
Functionally, decreases in ER protein levels can lead to decreases in E
responsiveness, as demonstrated by transfection studies in the present
study. Estrogen pretreatment elevated absolute luciferase expression
before E treatment, even though ER levels were reduced. It is
unlikely that ERs were saturated, because E treatment still led to a
2.5-fold increase in PRLluc expression. However, ICI182,780
pretreatment led to similar reductions in ER protein levels without
increasing basal reporter activity. The subsequent response to E was
the same as that with E pretreatment, suggesting that the reduction in
E responsiveness results from a reduction in ER expression rather than
an elevation in basal ER activity. Because TERP protein was
undetectable in GH3 cells, it is unlikely that
E-induced TERP expression contributed substantially to the reduced E
responsiveness of the PRLluc reporter in pretreated cells because TERP
levels must exceed those of full-length ERs to inhibit responsiveness
(12, 13).
ERß mRNA levels were lowest on the morning of proestrus, when
in vivo steroid levels are highest. Changes in steroid
levels probably mediate this effect in females because treatment of
ovariectomized animals with E, E plus P, or DHT suppressed ERß mRNA
expression. This is the first demonstration of steroid down-regulation
of ERß mRNA in the pituitary; however, Mitchner et al. (6)
observed a similar, but nonsignificant, effect in E-treated rats. ERß
mRNA down-regulation is also intriguing in light of recent evidence
that ERß expression in the female rat pituitary decreases after
puberty, again suggesting an effect of increased circulating steroids
(16). During the estrous cycle, this suppression of ERß mRNA could be
associated with a decrease in protein levels around the time of the
proestrous surge and, like ER suppression, would lead to a decrease
in E-mediated transcription. In contrast to our in vivo data
and a published report showing up-regulation of ERß by 24 h of E
treatment in GH3 cells (34), E and DHT had no
effect on ERß mRNA expression in pituitary cell lines. E and DHT
could affect the pituitary indirectly by altering the release of
hypothalamic factors, but we saw no direct evidence of regulation by
GnRH in cell lines or by GnRH or dopamine in vivo.
Alternatively, the effects on ERß in whole pituitary might be due to
direct or indirect effects on cell types other than lactotropes or
gonadotropes. During the estrous cycle, the fall in ERß precedes
increases in circulating P, and P alone did not alter ERß mRNA in
rats, GH3 cells, or RC4B cells in the absence of
E. Although this may be due to a requirement for E induction of PR, the
observation that P alone decreases ERß in LßT2 cells suggests that
P might have a role distinct from that of E. Two additional lines of
evidence support a role for P in ERß regulation. A recent report
demonstrated that P reduces ERß mRNA levels in T-47D breast cancer
cells, and an inverse correlation between ERß mRNA levels and PR
expression was observed in breast tumor biopsies (42).
In contrast to ER and ERß, TERP-1 mRNA began to rise on diestrus
and peaked on the morning of proestrus, when in vivo steroid
levels are highest, as previously demonstrated (8, 18). Estrogen
consistently stimulates TERP-1 mRNA expression in vivo (5, 6, 8, 17, 18), in GH3 cells (34), and in
pituitary cell lines in the present study. The E-induced increase in
TERP-1 mRNA levels is recapitulated in the expression of TERP-1 protein
in ovariectomized rats treated with E, in which TERP-1 protein levels
equal or exceed those of the full-length ER, and in our pituitary
cell lines, where TERP-1 protein can be detected. At the ratios
observed in vivo, TERP-1 inhibits ER-mediated transcription
(12), and the transcriptional activity of remaining ER and ERß at
proestrus would be further reduced.
Although the results from no one cell line completely describe events
in the intact rat pituitary gland, several regulatory pathways appear
to be identical between the pituitary and lactotrope and gonadotrope
cell lines. These include the decrease in ER protein with E
treatment, the decrease in ERß mRNA with E plus P treatment, and the
increase in TERP-1 mRNA and protein with E. TERP-1 has biphasic effects
on ER and ERß activity, stimulating ER at low ratios and
inhibiting activity at a ratio of 1:1. Thus, after proestrus, ER
protein and potentially ERß protein will be decreased, and TERP-1
protein will approach or exceed a ratio of 1:1 with full-length
receptors. Under these conditions, TERP can further suppress
ER-mediated transcription (12, 13). We hypothesize that increased TERP
expression in the face of decreased full-length ER levels effectively
suppresses E actions in the pituitary and may play a role in
terminating the proestrous surge.
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Acknowledgments |
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Footnotes |
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Received September 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
ß in the rat pituitary gland. Endocrinology 139:3976–3983. Mol Endocrinol 11:1486–1496 and ß. J
Biol Chem 275:7158–7166 and ß. Endocrinology 138:863–870 (ER-) and B
(ER-ß) mRNA in the rat pituitary, gonad, and reproductive tract.
Steroids 63:498–504[CrossRef][Medline]
(ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621
receptor chimeras. Endocrinology 139:4513–4522 and
ß isoforms in human pituitary tumors. J Clin Endocrinol Metab 83:3965–3972 and ß in GH3 cells. Endocrinology 140:2651–2658This article has been cited by other articles:
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