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and ß in GH3 Cells1
Department of Cell Biology, University of Cincinnati Medical School, Cincinnati, Ohio 45267
Address all correspondence and request for reprints to: Dr. Nira Ben-Jonathan, Department of Cell Biology, University of Cincinnati Medical School, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0521. E-mail: nira.ben-jonathan{at}uc.edu
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Abstract |
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(ER) and -ß (ERß). A truncated
isoform of ER, named TERP, is expressed in the pituitary, but not in
the uterus. In this study we used the somatolactotroph cell line,
GH3 cells, to examine 1) the expression of ER, TERP, or
ERß and their regulation by estradiol; 2) the presence of receptor
proteins; and 3) the effects of overexpressing ERß or TERP on
estrogen induction of the PRL gene and activation of the estrogen
response element (ERE).
Incubation of GH3 cells with estradiol (0.1–10
nM) produced dose-dependent increases in messenger RNA
levels of ERß and TERP, but not ER, as determined by quantitative
RT-PCR. Cell incubation with 1 nM estradiol resulted in a
time-dependent biphasic increase in TERP and a delayed rise in ERß,
suggesting activation by both direct and indirect mechanisms. A
polyclonal ERß antibody directed against an N-terminal synthetic
peptide was generated. This antibody detected ERß-positive cells in
ovarian granulosa cells and in many cells throughout the pituitary; its
specificity was demonstrated by preabsorption with the synthetic
peptide. The antibody detected a 58- to 60-kDa protein by Western
blotting of ovarian, pituitary, and GH3 cell extracts.
Cotransfection of ERß and reporter genes (PRL promoter/luciferase or
ERE/luciferase) into GH3 cells resulted in a dose-dependent
increase in estrogen-induced PRL gene expression, with a lesser
activation of the ERE. A 20-kDa TERP protein was undetectable in
untreated GH3 cells and was weakly induced by estradiol.
Overexpression of TERP had no effect on estrogen induction of either
PRL or ERE.
We conclude that 1) both ERß and TERP messenger RNAs in
GH3 cells are increased by estradiol in a dose- and
time-dependent manner, whereas ER is not altered; 2) a 58-kDa ERß
protein is expressed in both the pituitary and GH3 cells;
and 3) overexpression of ERß increases estrogen-induced PRL gene
expression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, its
truncated isoform, and ERß.
ERß, first identified in the rat prostate and ovary (9), shares
several similarities with ER. Both receptors have a high binding
affinity for estrogen and activate the same estrogen response element
(ERE) (10), but differ in their trans-activation domains,
suggesting distinct roles in gene activation. At the messenger RNA
(mRNA) level, ERß has been detected in many tissues, including rat
(11, 12) and human (13), but not mouse (14), pituitaries. Using a
combined immunocytochemistry/in situ hybridization approach,
we detected ERß expression in most pituitary cell types, including
30% of the lactotrophs (12). However, ERß has not been well
demonstrated at the protein level due to the limited availability of
specific antibodies. In addition, the specific actions of this receptor
within the pituitary gland have not been delineated.
The truncated estrogen receptor product (TERP), discovered by Shupnik
et al. (15, 16), lacks exons 1–4 of ER but contains most
of the hormone-binding domain and the second
trans-activation domain. Given these structural properties,
TERP may act either as a dominant negative regulator or as an enhancer
of estrogenic action. TERP is expressed in the pituitary, but not in
the uterus, and its mRNA levels increase in response to estrogen (12, 16). Pituitary TERP gene expression is altered during the estrous
cycle, with the highest mRNA levels seen on the morning of proestrus
(17). Although not all of the pituitary cell types that express TERP
have been identified, cell separation experiments revealed that the
lactotroph-enriched fraction had the highest amount of TERP mRNA
(18).
GH3 cells, a rat somatolactotroph cell line, are widely used as an in vitro model for the lactotrophs. When these cells are maintained under low estrogen conditions, exogenous estrogens increase PRL synthesis and release (19, 20, 21) and exert a biphasic effect on cell proliferation (1). Binding studies revealed the presence of ERs in GH3 cells (22), but neither the identity of the receptor nor its regulation by estrogen have been examined.
In this study, we used GH3 cells as an in vitro
model for investigating several aspects of the ERs in lactotrophs. The
objectives were to 1) examine the expression of ER, TERP, and ERß
and compare their regulation by estradiol; 2) generate an ERß
antibody to characterize ERß protein expression in the pituitary and
GH3 cells; and 3) determine the effects of ERß or TERP
overexpression on the PRL gene and ERE activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antiserum
antiserum (directed against a synthetic peptide at the extreme
C-terminus of ER) was provided by Dr. M. Shupnik (University of
Virginia, Charlottesvile, VA).
Animals
All animal studies were performed under an institutionally
approved protocol according to the USPHS Guide for the Care and Use of
Laboratory Animals. Intact and ovariectomized Fischer 344 rats (7–8
weeks old) were obtained from Zivic-Miller (Zelienpole, PA) and were
maintained under standard conditions.
GH3 cell culture
GH3 cells were maintained in Ham’s F-10 medium
supplemented with 15% heat-inactivated gelding serum (Central
Biomedia, Lenexa, KS) that has undetectable estrogen levels. Two or 3
days before an experiment, cells were plated in serum-free medium
composed of DMEM and Ham’s F-10 (50:50) supplemented with ITS+ premix
(Collaborative Research, Inc., Bedford, MA) and
penicillin/streptomycin. For RT-PCR, 500,000 cells were plated in
12-well plates coated with protamine (Sigma Chemical Co.,
St. Louis, MO) and Nu-Serum (Collaborative Research, Inc.)
as previously described (23). For transfection experiments, 75,000
cells were plated on similarly coated 24-well plates.
RT-PCR for ER, TERP, and ERß
Total RNA was isolated from GH3 cells using
Tri-Reagent (Molecular Research Center, Inc., Cincinnati,
OH), and 5 µg were reverse transcribed using random hexamers and
Superscript II reverse transcriptase (Life Technologies,
Gaithersburg, MD) as previously described (12). Primer sequences and
expected product sizes were: 1) ER sense primer,
5'-GGTCCAATTCTGACAATCGAGC-3'; ER antisense primer,
5'-TTTCGTATCCCGCCTTTCATC-3' (with an expected size of 304 bp); 2) TERP
sense primer, 5'-GCTTGTTGAACAGCGACCAG-3'; TERP antisense primer,
5'-CTTGTCCAGGACTCGGTG-3' (with an expected size of 366 bp for TERP1);
and 3) ERß sense primer, 5'-AACACTTGCGAAGTCGGCAG-3'; ERß antisense
primer, 5'-AACCTCAAAAGAGTCCTTGGTGTG-3' (with an expected size of 327
bp). Each reaction also contained primers for ribosomal protein L19
(RPL19), which served as an internal control: sense primer,
5'-AGTAGTCTTAGGCTACAGAAG-3'; and antisense primer,
5'-TTCCTTGGTCTTAGACCTGCG-3' (with an expected size of 500 bp).
Optimal PCR conditions for each receptor were determined by varying the cycle number, RNA concentration, and annealing temperature as previously described (12). PCR products were separated on a 1% agarose gel stained with ethidium bromide, photographed, and analyzed by scanning densitometry (Scion Image Software, Frederick, MD).
ERß antibody production
An 18-amino acid peptide from the N-terminus of ERß (amino
acids 48–65) was made by solid phase synthesis on a multiple antigenic
peptide backbone (Protein and Carbohydrate Structure Facilities,
University of Michigan, Ann Arbor, MI). New Zealand rabbits (Harlan
Bioproducts, Indianapolis, IN) were used for a standard 90-day
protocol. Enzyme-linked immunosorbent assays were performed to
determine antibody titer.
Immunohistochemistry for ERß
Immunohistochemistry was performed as previously described (5).
Briefly, pituitary or ovary cryosections (10 µm) were mounted on
silane-coated slides and fixed in 4% paraformaldehyde for 20 min.
Endogenous peroxidase activity was blocked with 3% hydrogen peroxide
in 70% methanol for 30 min at 4 C, and the sections were permeabilized
with 0.1% Triton-X for 10 min. The slides were incubated with ERß
antiserum (1:1000) overnight at 4 C, and staining was performed using
biotinylated secondary antibodies and avidin-biotin-peroxidase complex
(ABC kit, Vector Laboratories, Inc., Burlingame, CA).
Brown color was visualized after 2-min incubation with
diaminobenzidine. Some sections were counterstained with hematoxylin.
For controls, 50 µl of the antiserum were preabsorbed with 100 µg
of the synthetic peptide by overnight incubation at 4 C. Both antibody
and synthetic peptide are available from Upstate Biotechnology (Lake
Placid, NY).
Western blotting for ER, TERP, and ERß
Pituitaries and ovaries were removed and immediately frozen.
GH3 or COS-1 cells were pelleted and frozen. Samples were
homogenized in lysis buffer [50 mM Tris-HCl (pH 7.5), 150
mM NaCl, and 5 mM EDTA] containing the
following protease inhibitors: 0.25% sodium deoxycholate, 1
mM phenylmethylsulfonylfluoride, 1 mM NaF, and
1 µg/ml each of aprotinin, leupeptin, and pepstatin. Homogenates were
centrifuged at 13,000 x g, and the supernatants were
subjected to protein determination with a BCA protein assay
(Pierce Chemical Co., Rockford, IL). Recombinant human
ERß (100 ng) from PanVera (Madison, WI) served as a positive control.
Proteins (30 µg) were electrophoresed on a 10% or 12%
polyacrylamide gel, electrotransferred to nitrocellulose membranes
(Schleicher & Schuell, Inc., Keene, NH), and stained with
Ponceau S (Sigma Chemical Co.) to visualize the transfer.
The blots were blocked with 2% nonfat milk and incubated overnight at
4 C with either ERß (1:2000) or ER (1:7500) antisera. An enhanced
chemiluminescence kit (Amersham, Arlington Heights, IL)
was used to visualize the products.
Subcloning of TERPs
TERP expression vectors using alternative start sites (see Fig. 6
) were PCR amplified from anterior pituitary complementary DNAs
(cDNAs) using the following primers: Met401 sense primer,
5'-TTGAATTCGATTGGTCTGGTCTG-3'; Met426 sense primer,
5'-TTGAATTCGGACAGGAATCAAGG-3'; and the same antisense primer for both,
5'-TTTCTAGAGCTTCTCAGATGGTGTT-3'. The products were ligated into a
pTarget expression vector (Promega Corp., Madison, WI) and
confirmed by sequence analysis.
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COS-1 cells, maintained in DMEM and 10% FBS, were trypsinized, and 1 x 106 cells were plated on 100-mm plates. For transfection, 5 µg of each of the different TERP expression vectors and 35 µg lipofectamine were incubated for 6 h, followed by the addition of medium containing gelding serum for 24 h.
Reporter gene assays
GH3 cells were washed in PBS and lysed in 100 µl
lysis buffer (Promega Corp.), and duplicate 20-µl
aliquots were transferred to separate black plates (Packard Instrument
Co., Meriden, CT). For luciferase determination, 80 µl luciferin
substrate (Promega Corp.) were added. For
ß-galactosidase determination, 80 µl Galacton-Plus substrate
(Tropix, Bedford, MA) were added and incubated for 25 min at room
temperature, and then 200 µl of accelerator II (Tropix) were added
immediately before counting. Luminometry was quantitated by using a
Packard TopCount.
Data analysis
For RT-PCR, the density ratio of receptor/RPL19 bands was
calculated and expressed as a percentage of the control values (no
estrogen), with each data point representing four to six
determinations. For transfections, the ratio of reporter
gene/ß-galacosidase activity was determined and expressed as a
percentage of control values (no estrogen). Triplicate transfections
were performed four to six times. All values are expressed as the
mean ± SEM. Statistical significance was determined
using ANOVA followed by Dunnett’s post-hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TERP, and ERß mRNA levels are
shown in Fig. 1, left panel.
As determined by quantitative RT-PCR, the expression of TERP increased
in a dose-dependent manner, reaching levels 4- and 5-fold above control
values in response to 1 and 10 nM estradiol, respectively
(P < 0.05). ERß mRNA levels increased 3-fold by 1
nM estradiol (P < 0.05) without a further
rise with the 10-nM dose. In contrast, ER mRNA levels
were unaffected by estradiol at all doses tested. A dose of 1
nM estradiol was selected for the time-course experiments.
As shown in Fig. 1, right panel, estrogen treatment
increased both TERP and ERß mRNA levels in a time-dependent manner,
whereas ER levels were unchanged. Interestingly, TERP mRNA levels
rose in a biphasic manner, reaching 6-fold above control values at
3 h (P < 0.05), decreasing to 3-fold by 6 h
(P < 0.05), and rising again at 24 h
(P < 0.05). Compared with TERP, the rise in ERß mRNA
was delayed, reaching its highest level of 3-fold above the control
values (P < 0.05) after 24 h.
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, upper left
panel, shows a mature Graffian follicle positively stained for
ERß protein, with a weaker and more diffuse staining seen in the
ovarian stroma as well as near blood vessels. The two insets
demonstrate strong brown nuclear staining in the majority of granulosa
cells surrounding the ovum and the antral cavity. The marked
attenuation of nuclear staining by ERß antisera preabsorbed with the
synthetic peptide (upper right panel) establishes the
specificity of the antibody.
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, lower left panel, shows the distribution of
ERß-positive cells in the pituitary gland. Nuclear staining for ERß
was observed in many cells within all lobes of the pituitary, with a
high concentration of ERß-positive cells at the interface between the
anterior and intermediate lobes. The left inset, showing an
enlargement of a region within the anterior lobe, reveals that less
than 50% of the cells are ERß positive. Immunostaining in the
absence of hematoxylin is shown in the right inset. Again,
antibody specificity was demonstrated by preabsorption (lower
right panel).
ERß protein is expressed in both the pituitary and
GH3 cells
Western blotting was performed on extracts of ovary, pituitary,
and GH3 cells to establish the presence of ERß, confirm
its size, and support the histological validation. Figure 3 shows a protein band of 58–60 kDa,
calculated from molecular mass markers, in all three tissues. The same
electrophoretic mobility is exhibited by a recombinant human ERß
protein. A 35-kDa band, which may be nonspecific, was also observed in
some of the extracts.
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, left panel,
18 h of estradiol treatment of cells transfected with an empty
vector increased PRL gene expression 3-fold over the control level.
When increasing amounts of ERß were introduced into the cells, PRL
gene expression was enhanced in a dose-dependent manner, reaching
6-fold above the control level with 25 ng ERß (P <
0.05). On the other hand, overexpression of ERß, at the highest
amount tested, only slightly activated a triple vitellogenin ERE (Fig. 4, right panel).
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(66-kDa) and TERP (20-kDa) proteins were detected in the
pituitary, only ER was found in untreated GH3 cells
(Fig. 5). A very weak 20-kDa protein band
(evident only after overexposure of the blot) was seen in
GH3 cells incubated with 1 nM estradiol for
24 h. To examine whether GH3 cells can translate an
exogenous TERP, the cells were transfected with a TERP expression
vector (gift from Dr. Friend) (24). As shown in Fig. 5, the transfected
cells showed three protein bands, the wild-type ER, a 55-kDa protein
of an unknown identity, and a 24-kDa protein, presumably the
transfected TERP.
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) led us to consider alternative translation
initiation sites of TERP. The exon 5 sequence contains three putative
Met start sites: 393, 401, and 426 (Fig. 6). When the original TERP expression
vector, which uses Met393, was transfected into COS-1
cells, the resulting 24-kDa protein was larger than the endogenous
pituitary TERP (Fig. 6); the same size protein was also seen in
transfected GH3 cells (Fig. 5). The cDNAs with the two
other potential start sites were PCR cloned and expressed in COS-1
cells. As evident, the Met426 is probably used by the
pituitary as the initiation site for TERP translation. This vector,
which also produces a 20-kDa protein in GH3 cells (data not
shown), was used for further experiments.
TERP overexpression does not alter the expression of selected
reporter genes
GH3 cells were cotransfected with the TERP vector and
PRL/luc or ERE/luc reporter genes, using the same
paradigm as that described for ERß. Figure 7, left panel, shows that the
TERP vector, at all concentrations examined, did not alter the
estrogen-induced PRL gene expression above that obtained with an empty
vector. Similarly, ERE reporter activity was unchanged by TERP
overexpression (right panel). Further increases in the
amount of TERP expression vector resulted in a marked decrease in the
transfection efficiency, presumably because of a lower cell
survival.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TERP, and ERß. Estrogen induces the expression of both ERß
and TERP, but not ER, in these cells. Our novel ERß antibody
identifies a significant number of immunopositive cells in all three
lobes of the pituitary and detects a 58-kDa protein band in both the
pituitary and GH3 cell extracts. Overexpression of ERß
significantly enhances estrogen-induced PRL gene expression, but is
less effective in activating an ERE construct. On the other hand,
overexpression of TERP does not alter either PRL or ERE in response to
estrogen. Both TERP and ERß mRNA levels in GH3 cells were up-regulated by exogenous estrogen in a time- and dose-dependent manner. The increase in TERP was biphasic, suggesting that the rapid 5-fold rise was due to a direct transcriptional effect of estrogen, whereas the delayed increase may represent a secondary effect by induced proteins. The delayed increase in the ERß mRNA levels appears to be due to a secondary response. In our previous in vivo study, estrogen induced an increase in anterior pituitary TERP mRNA levels, but did not alter ERß (12). As the pituitary contains different estrogen-responsive cells, including lactotrophs, gonadotrophs, and folliculo-stellate cells (5, 25, 26), specific changes in each cell type cannot be distinguished using the whole pituitary. In fact, we have estimated that only 30% of the pituitary lactotrophs express ERß mRNA (12). The up-regulation of ERß gene expression by estrogen in GH3 cells suggests that ERß in lactotrophs is sensitive to estrogen.
To examine ERß at the protein level, we generated polyclonal antibodies and used both immunohistochemistry and Western blotting for their characterization. Strong nuclear staining was seen in ovarian granulosa cells, as expected from in situ hybridization studies (9, 12, 27), with the staining specificity confirmed using preabsorbed antisera. Cells with nuclear staining for ERß were also observed in all lobes of the pituitary, in agreement with the distribution of ERß mRNA-expressing cells in the pituitary (12). Furthermore, the antibody detected a 58- to 60-kDa ERß protein in ovary, pituitary, and GH3 cells. Although this protein differs slightly from the 54 kDa predicted size of the original cDNA clone (9), several ERß isoforms have been reported (28, 29).
After demonstrating that GH3 cells express ERß, we
examined whether it mediates the actions of estrogen on the PRL gene.
After estrogen binds to its receptor (either ER or ERß), the
receptor dimerizes and binds to an ERE consensus sequence in the
promoter region of responsive genes. An imperfect palindromic ERE is
located in the distal promoter region of the PRL gene, adjacent to a
pit-1 site. Pit-1, a pituitary-specific transcription factor, is
mandatory for estrogen induction of the PRL promoter (30, 31). Hence, a
cell line such as GH3 with an endogenous pit-1 is an
appropriate model for studying the effect of estrogen on the PRL
gene.
The cotransfection experiments were designed to overexpress ERß and
examine its action on both the PRL promoter and the vitellogenin ERE.
As shown in Fig. 4, transfection with increasing amounts of ERß
resulted in a dose-dependent rise in estrogen-induced PRL gene
expression, with a lesser activation of the ERE. A synergism between
the activated receptor and pit-1 may explain the higher responsiveness
of the PRL gene to estrogen than the transfected ERE. These experiments
could not discriminate between homodimerization of ERß or its
heterodimerization with ER, as was recently reported in
vitro (10). Experiments are underway to dissect out the roles of
ER and ERß by selectively reducing the expression of each
receptor. Unfortunately, the role of ERß in pituitary physiology
cannot be studied using the ER-deficient mouse (32), because the
mouse pituitary does not express ERß (14) (our unpublished
observations).
Among the many ER variants identified at the mRNA level (33), TERP
appears to be the only variant shown to be translated. Therefore, it
was of interest to examine the functions that might be mediated by this
truncated receptor. In the process of preparing a TERP expression
vector, we identified Met426 as the likely pituitary
translation start site, based on its size similarity to the endogenous
TERP protein. This methionine is flanked by the consensus initiation
sequence, A/GNNATGG (34). As TERP can bind estrogen and contains the
domains for coactivator and receptor dimerization, it could suppress
estrogenic action by squelching either exogenous estrogen or the
wild-type receptor, or it may enhance transcription by recruiting
receptor coactivators (35). In human 293 kidney cells, transfected TERP
was reported to increase ER-mediated activation of a vitellogenin
ERE (24). An N-terminal-truncated progesterone receptor was also shown
to enhance transcription from a mouse mammary tumor virus promoter
(36). However, we were unable to demonstrate an effect of TERP
overexpression in GH3 cells on either PRL or ERE. Whereas
endogenous TERP protein is detectable in the pituitary (12, 17), only a
very weak expression is evident in estrogen-treated GH3
cells. Clearly, additional studies are needed to elucidate the role, if
any, of TERP in pituitary function.
In summary, we have established that GH3 cells,
representing the pituitary lactotrophs, express ER, ERß, and TERP.
The precise role of each of these receptors and whether they have
distinct, overlapping, or synergistic functions remain to be
determined. In addition to PRL, the lactotrophs have other
estrogen-regulatable genes, and it would be important to identify which
receptor mediates their regulation.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Pediatrics, Indiana University School
of Medicine, Indianapolis, Indiana 46202.
Received October 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Mol Endocrinol 11:1486–1496 and ß. Endocrinology 138:863–870 and
ß in the rat pituitary gland. Endocrinology 139:3976–3983) and beta
(ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol
Metab 82:3509–3512 (ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
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