| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Medical Research Laboratory, Wolfson Building, University of Hull, Hull HU6 7RX, United Kingdom
Address correspondence and requests for reprints to: Dr. Valerie Speirs, Molecular Medicine Unit, Clinical Sciences Building, St. James’s University Hospital, Leeds LS9 7TF, United Kindgom. E-mail: v.speirs{at}leeds.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, wild-type ERß (mRNA and protein), and ERß exon 5 deletion
variants (ERß 
5) in samples of normal human mammary gland obtained
from 37 premenopausal subjects undergoing reduction mammoplasty.
Comparing individual expression, ERß mRNA predominated, expressed in
34 of 37 samples (91%), whereas ER was found in 21 of 37 cases
(57%). Receptor combinations were then analyzed and compared. Most
samples either coexpressed ER with ERß (54%) or expressed just
ERß (38%). Immunohistochemical analysis revealed that ERß mRNA
expression mirrored that of protein. Immunoreactivity was observed in
the nucleus with additional evidence of cytoplasmic staining in those
epithelial cells lining the breast ducts. Sporadic immunoreactivity was
also detected in stromal cells. Expression of wild type and ERß 5
was analyzed, and their association with ER was compared. Most
samples coexpressed wild-type ERß and the splice variant (62%;
P = 0.05), with 30% exclusively expressing
wild-type ERß. Although samples coexpressing wild type and variant
ERß showed no statistical association with ER, those samples
expressing only wild-type ERß, showed a trend toward associations
with ER (P = 0.07). In conclusion, our data
would support a role for ERß in the normal human mammary gland, where
we propose it may be the dominant receptor. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) in 1986 (1, 2), it was believed only a single
receptor existed. This was in contrast to other members of the
superfamily of steroid-thyroid-retinoic acid receptors in which
multiple family members are found (3). Ten years after the cloning of
ER, steroid receptor biochemistry entered a new chapter with the
cloning of a second ER, known as ERß, initially from rodent and later
from human tissues (4, 5, 6). Both receptors show high homology at the
DNA- and ligand-binding domains (96% and 58%, respectively), whereas
the A/B, hinge, and F-regions are not well conserved (6). The genes for
both receptors are encoded by eight exons, although these are located
on different chromosomes, ER on the long arm of chromosome 6 and
ERß on chromosome 14q22–24 (7), confirming that each receptor is the
product of independent genes.
Differences in tissue distribution and relative expression of messenger
RNA (mRNA) for both receptors have been described, with altered
expression associated with carcinogenesis in both breast and ovary
(8, 9, 10). Recently, we have shown that in normal breast expression of
ERß predominated, with exclusive expression of this receptor quite
common, a feature not observed in a cohort of breast tumors (11).
Furthermore, the relatively small number of cells (up to 15%) reported
to express ER protein in premenopausal breast tissue (12, 13, 14) raises
the question that ERß may be significant in the normal human mammary
gland.
Additional ER mRNA isoforms, generated by alternative mRNA splicing,
have been described in many tissues, including the breast. ER
variant mRNAs are relatively common in breast tumors (15) but are also
expressed in normal breast where five different exon-deleted variants
(exons 2, 3, 2–3, 5, and 7) and one truncated variant (clone 4) have
been described (16). However, the detection of ERß variants in the
mammary gland has so far been restricted to two studies, which have
provided conflicting results. Vladusic et al. (17) reported
that expression of exon 5-deleted variant (ERß 5) was confined to
breast tumors and not seen in normal breast, whereas exons 5, 6, or
5–6 deletions have been identified both in breast tumors and a small
number of normal human mammary tissues (18).
The aims of this study were 3-fold. First, we analyzed and compared
expression of ER and ERß mRNA in normal mammary gland. Next, we
focused on ERß; the majority of published studies on ERß have
presented data on gene rather than protein expression, and we sought to
resolve whether gene expression paralleled that of protein using
immunohistochemistry. Finally, we determined whether exon 5 deletion
variants of ERß are expressed in normal breast and whether there were
any associations with expression of ER.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Normal human breast tissue was obtained from 37 premenopausal subjects who presented sequentially for reduction mammoplasty and who had no previous history of breast disease (mean age, 35 yr; range, 18–42). Ethical approval was obtained, and all patients gave informed consent. Tissue was trimmed of excessive adipose tissue and snap-frozen on receipt and stored at -80 C until required. Frozen tissue was pulverized using a mortar and pestle, and total RNA was extracted with Trizol (Life Technologies, Inc., Paisley, UK) according to the manufacturer’s instructions. RNA (1 µg) was used as a template for first strand synthesis, as described previously (19).
PCR amplification
Primers were obtained from Life Technologies, Inc.
and were designed from published gene sequences. Primer sequences and
reaction conditions for ER have previously been published (19). To
detect wild-type ERß and ERß 5, a nested PCR was performed as
described (17), except that 5 µl complementary DNA (cDNA) was
initially amplified. One microliter of resultant PCR product was
removed and reamplified using a second set of primers (17). Both PCR
reactions contained: 2 U BioTaq; 10x PCR buffer (containing 1.5
mM MgCl2; both from Bioline, London,
UK); 0.5 µg of each oligonucleotide primer; 200 µM each
of dATP, dCTP, dGTP, and dTTP (Roche Molecular Biochemicals, East Lewes, UK); 5 µl nascent cDNA (PCR 1) or 1
µl PCR product from reaction 1 (PCR 2); and sterile distilled water
to bring the volume to 50 µl. To check cDNA integrity, fragments of
glyceraldehyde-3-phosphate dehydrogenase, a standard housekeeping gene,
were amplified in parallel; this was consistently positive (data not
shown). As a positive control for ER, cDNA from the human breast
cancer cell line MCF-7 was used; for ERß, human testis cDNA was used.
Negative controls included substitution of RNA or cDNA with distilled
water, or substitution of cDNA with an irrelevant cDNA. These were
consistently negative. All transcripts were analyzed in parallel on at
least two separate occasions in a thermal cycler (Hybaid OmniGene,
Teddington, UK). PCR products were analyzed by electrophoresis through
a 1.2% agarose gel and visualized by ethidium bromide staining under
ultraviolet illumination.
Immunohistochemistry
Cryostat sections (6–7 µ) of snap-frozen breast tissue were
prepared from 10 individual cases and mounted onto polylysine-coated
slides. Slides were air dried, fixed in absolute methanol for 10 min at
room temperature, then rehydrated in phosphate-buffered saline.
Sections were then incubated for 5 min in 3% hydrogen peroxide to
block endogenous peroxide and incubated overnight at 4 C with an
affinity-purified goat polyclonal antibody directed against a peptide
from the N terminus of human ERß (N-19; Autogen Bioclear, Wiltshire,
UK; 1:100 dilution). This antibody shows no cross-reactivity with
ER. To confirm the specificity of the antibody, it was neutralized
by incubation with a 5-fold excess of blocking peptide [ERß (N19) P;
Autogen Bioclear] for 2 h at room temperature. Neutralized
antibody was included as a negative control in all experiments.
Positive ERß staining was detected using the ABC method (Vector Quick
Kit; Vector Laboratories, Inc. Peterborough, UK) with
diaminobenzidine as a substrate. Slides were lightly counterstained
with eosin, dehydrated through graded alcohols, cleared in xylene, then
mounted.
Cloning and sequencing of PCR products
PCR products were purified using Wizard DNA clean-up columns and
ligated into the pGEMT Easy PCR cloning vector (both from Promega Corp., Chandlers Ford, UK). Ligated plasmids were then
electroporated into competent DH5 Escherichia coli using
a Gene Pulser (Bio-Rad Laboratories, Inc., Hemel
Hempstead, UK). DNA was prepared from transformed cells using the
Wizard SV mini-prep system. Three different plasmid clones were
sequenced (ALF Express; Amersham Pharmacia Biotech, Little
Chalfont, UK), using DNA prepared with a 7-deaza dGTP cycle sequencing
kit (Amersham Pharmacia Biotech).
Statistical analysis
Statistical analysis was performed using the Arcus software
package for Windows (Research Solutions, Cambridge, UK). Fisher’s
exact test was used to test the difference between groups. Results were
considered to be significant at P 
0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
By RT-PCR, both ER and ERß were detected. When their
individual expression was considered, ERß predominated, expressed in
34 of 37 samples (91%). ER was observed in 21 of 37 cases (57%),
whereas 2 of 37 samples failed to express either receptor, perhaps
indicting the presence of a third receptor. Receptor combinations were
then analyzed and compared. The results are summarized in Table 1
. Most samples either coexpressed ER
with ERß or expressed just ERß. To determine whether gene
expression paralleled that of protein, sections from 10 normal mammary
gland biopsies, shown to be ERß positive by RT-PCR, were
immunostained with an antibody directed against ERß. Gene
expression mirrored protein expression in all cases. ERß
immunoreactivity was predominantly restricted to the nucleus and
cytoplasm of those epithelial cells lining the breast ducts with more
limited evidence of focal positivity in the surrounding stromal tissues
(Fig. 1).
|
|
5 in normal human mammary gland
Because wild-type ERß gene and protein was detected in the
normal human mammary gland, we next investigated whether ERß 5
mRNA was present. A representative agarose gel showing transcripts for
wild-type ERß (429 bp) and a smaller exon 5 deletion variant (290 bp)
is shown in Fig. 2. Sequence analysis
confirmed that the smaller PCR fragment contained a 139-bp deletion
(nucleotides 812–950), corresponding to the entire exon 5 of human
ER (similarity = 99%, identity = 98%, data not shown).
Expression of wild type and variant ERß was analyzed, and their
association with ER was compared. As detailed in Table 2, a significantly higher proportion of
samples coexpressed wild-type ERß with ERß 5 (62%;
P = 0.05) with fewer samples exclusively expressing
wild-type ERß. Furthermore, ERß 5 was detected only in
combination with wild-type ERß. Samples that coexpressed wild type
and variant ERß showed no statistical association with ER.
However, in samples exclusively expressing wild-type ERß, there was a
suggestion of a trend toward associations with ER (P
= 0.07). In two cases, tissue was available from both breasts. These
gave identical profiles, indicating that breast tissue from the same
woman expresses the same ER profile (data not shown).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and
ERß. Using RT-PCR analysis of total RNA from normal human mammary
gland, we have shown expression of wild-type ERß and ERß 5.
Furthermore, by immunohistochemistry, we have demonstrated that
expression of wild-type ERß mRNA parallels protein expression.
Comparing individual mRNA expression for each receptor subtype, ERß
appeared to be the dominant receptor, observed in 91% of samples,
whereas ER was found in 54%, with coexpression of both receptors in
an equivalent number of cases. Immunohistochemical studies have
confirmed the presence and distribution of ER in epithelial cells
from normal premenopausal mammary gland. In some studies, this is
irrespective of the phase of the menstrual cycle (12, 20), whereas
others have revealed a higher proportion of ER-positive cells in the
follicular phase (up to 15%), falling to 5% in the
luteal phase (21, 22). Immunohistochemical data on ERß have so far
been more limiting, possibly reflecting the difficulties in raising
specific human antibodies against this protein. ERß immunoreactivity
has been observed in multiple rat tissues, including cells of the
ovary, testes, prostate, and paraventricular/supraoptic nuclei (23, 24). There is scant information on its distribution in human tissues,
although two recent studies have reported ERß immunoreactivity in
bone (25) and breast (26) tissues. In the bone study, immunoreactivity
was observed in nuclear extracts of human osteosarcoma cells and in
sections of human bone where positivity was seen in osteoblast nuclei
(25). In the breast study, Western blot analysis showed constitutive
expression of ERß protein in breast cancer cell lines and expression
in three of five breast tumor biopsies (26). Using frozen sections, we
demonstrated ERß immunoreactivity in normal human breast. This was
seen in the nucleus of those epithelial cells lining the breast ducts,
with more limited evidence of cytoplasmic staining. Its predominant
nuclear localization closely parallels the tissue distribution of
ER, and the cytoplasmic staining concurs with the pattern of
staining observed in human osteoclasts (25). Unexpectedly, we also
observed focal ERß immunoreactivity in stromal cells, and the
presence of ERß in these cells has been confirmed by RT-PCR analysis
of enriched stromal cell cultures (data not shown). At present, the
significance of ERß immunoreactivity in stromal cells is unclear.
Also, it is not known whether the distribution of ERß varies
throughout the menstrual cycle, but these aspects should be the focus
of future studies.
In normal breast it has been proposed that epithelial cells are
hierarchical in organization, with proliferation of
ER- cells under the control of paracrine
factors released from their ER+ counterparts
(14). However, the demonstration of ERß immunoreactivity suggests
that distribution/colocalization of both receptors may be relevant as
those cells thought previously to be ER()-
may express ERß. Our observation that a higher proportion of samples
expressed ERß is in contrast to a recent in situ
hybridization study of breast tumors, where ER was expressed in 72%
of samples compared with 44% of samples that expressed ERß (26).
This would support specific roles for each receptor in normal
vs. malignant breast where levels of expression may alter in
the evolution of breast cancer (8, 9, 10, 11).
Because ERß was clearly expressed in mammary gland, we investigated
whether ERß mRNA variants, specifically exon 5 variants, were also
found. Although our results contrast those of Vladusic et
al. (17), who only detected wild-type ERß mRNA in normal breast
tissue, this may be explained by sample size. Those investigators only
analyzed a single normal breast sample using mRNA from a commercial
source and two normal breast-derived organoid samples; our data show
that deletion variants are not constitutively expressed. Our results,
however, concur with Lu et al. (18), who also observed wild
type and variant ERß in normal human uterus, ovary, and mammary
gland. ERß 5 was coexpressed with wild-type ERß in 62% of
samples, but it was not expressed alone. If translated in
vivo, this exon-deleted mRNA would encode ER-like protein but
which lacks exon 5, part of the ligand-binding domain (7). Although
ligand-binding affinity may be lost or altered in ERß 5, it is
known that ER exon 5-deletion variants possess ligand-independent
activity in yeast expression systems (28). Because the DNA-binding
domain should remain intact in ERß 5 and would, therefore, still
be expected to form ERß homodimers or ER/ERß heterodimers (as a
result of coexpression of ER), this would permit interaction with
estrogen response elements and subsequent transcriptional activation of
target genes (29, 30). Furthermore, it has recently been shown that
ER/ERß heterodimers can be activated even if only one of the
cooperating partners binds ligand (31).
It is worth commenting that breast tissue from reduction mammoplasties may not be truly representative of normal breast tissue, and it would be of interest to analyze breast tissue from age-matched women without enlarged breasts. However, ethical considerations mean that such tissue is difficult to obtain. In some studies, tissue adjacent to breast tumors has been used as a source of normal tissue (8), but it is questionable whether this tissue is entirely normal, given the phenotypic and genotypic changes that occur during tumor development; some of these changes must surely influence the surrounding tissue milieu. It should, however, be noted that the reduction mammoplasty sections used in this study were examined by a pathologist, who confirmed that normal tissue architecture was maintained.
Although ERß 5 was clearly detected in over 60% of all normal
samples, at present its functional significance is unknown. Studies are
currently underway to compare the relative expression of ERß 5 in
premenopausal breast tumors with that of normal breast, however, no
significant differences in relative expression have yet been observed
in the small number of tumor samples analyzed, to date (data not
shown). This is in direct contrast to ER 5 where higher levels
are expressed in breast tumors (16). Although more detailed studies are
required, this would support the hypothesis that levels of expression
of each receptor subtype may differ in normal and malignant breast
(8, 9, 10, 11). In conclusion, our data would endorse a role for ERß in the
normal human mammary gland, possibly as the dominant receptor.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received July 1, 1999.
Revised August 31, 1999.
Accepted December 15, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß messenger RNA expression during
human breast tumorigenesis. Cancer Res. 58:3197–3201. and -ß messenger RNAs as a
potential marker of ovarian carcinogenesis. Cancer Res. 58:5367–5373. and ß: poor prognostic factors
in human breast cancer? Cancer Res. 59:525–528. and
progesterone receptor in relation to proliferating cells in the mammary
gland. Breast Cancer Res Treat. 53:217–227.[CrossRef][Medline]
and ß in human breast carcinoma. J Clin
Endocrinol Metab. 84:781–785. and ß form heterodimers on DNA. J Biol
Chem. 272:19858–19862.. J Biol Chem. 272:25832–25838.-ß heterodimeric complex. Mol Cell Biol. 19:1919–1927.This article has been cited by other articles:
 |
|
 |
|
  O. Treeck, G. Pfeiler, D. Mitter, C. Lattrich, G. Piendl, and O. Ortmann Estrogen receptor {beta}1 exerts antitumoral effects on SK-OV-3 ovarian cancer cells J. Endocrinol., June 1, 2007; 193(3): 421 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Ohnemus, M. Uenalan, J. Inzunza, J.-A. Gustafsson, and R. Paus The Hair Follicle as an Estrogen Target and Source Endocr. Rev., October 1, 2006; 27(6): 677 - 706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J-R Long, H Xu, L-J Zhao, P-Y Liu, H Shen, Y-J Liu, D-H Xiong, P Xiao, Y-Z Liu, V Dvornyk, et al. The oestrogen receptor {alpha} gene is linked and/or associated with age of menarche in different ethnic groups J. Med. Genet., October 1, 2005; 42(10): 796 - 800. [Full Text] [PDF]
|
|
|
|
 |
|
 C. Palmieri, E. W.-F. Lam, J. Mansi, C. MacDonald, S. Shousha, P. Madden, Y. Omoto, A. Sunters, M. Warner, J.-A. Gustafsson, et al. The Expression of ER{beta}cx in Human Breast Cancer and the Relationship to Endocrine Therapy and Survival Clin. Cancer Res., April 1, 2004; 10(7): 2421 - 2428. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Zheng, W. Zheng, B.-l. Chang, X.-O. Shu, Q. Cai, H. Yu, Q. Dai, J. Xu, and Y.-T. Gao Joint Effect of Estrogen Receptor {beta} Sequence Variants and Endogenous Estrogen Exposure on Breast Cancer Risk in Chinese Women Cancer Res., November 15, 2003; 63(22): 7624 - 7629. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Xu, Q. Pan-Hammarstrom, A. Forsti, K. Hemminki, L. Hammarstrom, D. Labuda, J.-A. Gustafsson, and K. Dahlman-Wright Human Estrogen Receptor {beta} 548 Is Not a Common Variant in Three Distinct Populations Endocrinology, August 1, 2003; 144(8): 3541 - 3546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V Speirs, G P Skliris, S E Burdall, and P J Carder Distinct expression patterns of ER{alpha} and ER{beta} in normal human mammary gland J. Clin. Pathol., May 1, 2002; 55(5): 371 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. LaVoie, D. C. DeSimone, C. Gillio-Meina, and Y. Y. Hui Cloning and Characterization of Porcine Ovarian Estrogen Receptor {beta} Isoforms Biol Reprod, March 1, 2002; 66(3): 616 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Saji, H. Sakaguchi, S. Andersson, M. Warner, and J.-A. Gustafsson Quantitative Analysis of Estrogen Receptor Proteins in Rat Mammary Gland Endocrinology, July 1, 2001; 142(7): 3177 - 3186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Cardenas, K.A. Burke, R.M. Bigsby, W.F. Pope, and K.P. Nephew Estrogen Receptor {beta} in the Sheep Ovary During the Estrous Cycle and Early Pregnancy Biol Reprod, July 1, 2001; 65(1): 128 - 134. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
