Full text of DOI:10.1359/jbmr.2001.16.2.214

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 2, 2001
© 2001 American Society for Bone and Mineral Research
Localization of Estrogen Receptor ␤ Protein Expression in
Adult Human Bone
ISOBEL P. BRAIDMAN,1 LINDA HAINEY,1 GAURAV BATRA,1 PETER L. SELBY,1
PHILIPPA T.K. SAUNDERS,2 and JUDITH A. HOYLAND1
ABSTRACT
Evidence suggests that the newly described estrogen receptor ␤ (ER-␤) may be important for estrogen
(17␤-estradiol) action on the skeleton, but its cellular localization in adult human bone requires clarification.
We addressed this by using indirect immunoperoxidase with a novel affinity purified polyclonal antibody to
human ER-␤, raised to hinge domain (D) sequences from the human receptor. Bone was demineralized in 20%
EDTA and all biopsy specimens were formalin-fixed and wax-embedded. Vigorous retrieval was essential for
ER-␤ detection. In sections (5 ␮m) of benign prostate hyperplasia, used as positive control, clear nuclear
immunoreactivity was seen in glandular epithelial cells, with a 1:500 dilution of ER-␤40. For bone sections,
optimal antibody dilutions were 1:100–1:250. We found that in normal bone (from graft operations), in
fracture callus from both men and women (>25 years old), pagetic bone, osteophytes, and secondary
hyperparathyroid bone, all from older patients, ER-␤ was expressed clearly in osteoclast nuclei, with little
cytoplasmic immunoreactivity. Nuclear immunoreactivity was still prominent in osteoclasts, with antibody
diluted 1:500, although it faded in other cells. Osteoblasts, in areas of active bone formation or bone
remodeling, also expressed ER-␤, as did some osteocytes. However, hypertrophic chondrocytes were negative,
unlike mesenchymal cells, adjacent to the osteogenesis. Megakaryocytes and some capillary blood vessels cells
were receptor positive. All ER-␤ expression was blocked totally by preincubation of antibody with antigen. We
conclude that ER-␤ is expressed in cells of osteoblast lineage and in osteoclasts. The latter appear relatively
abundant in this receptor and this might provide a means for direct action of estrogen on osteoclasts. (J Bone
Miner Res 2001;16:214–220)
Key words: estrogen receptor ␤, osteoclasts, osteoblasts, chondrocytes, indirect immunoperoxidase
fore, estrogen may be important to the skeletal development
and maintenance of bone mass in men as well as women.
Estrogen exerts its effects on target cells by interaction with
specific estrogen receptors (ERs) of which there are now
known to be at least two: ER-␣, which is expressed in
classic estrogen target cells, for example, in reproductive
tissues, and the newly described ER-␤,(5,6) the presence of
INTRODUCTION
HE IMPORTANCE of estrogen (17␤-estradiol) for maintain-
ing skeletal structure and function is well recognized in
women; falling estrogen levels in postmenopause are asso-
ciated with bone loss and increased risk of osteoporosis.
Moreover, in men who are deficient in the aromatase en-
T
zyme, because of gene mutation, there is failure of epiph- which accounts for the estrogen responsiveness of some
yseal fusion and severe osteopenia,(1,2) and falling estrogen tissues, despite their low ER-␣ expression.(7) Thus,
levels in aging men are associated with bone loss.(3,4) There- prostate,(8–10) ovary,(11,12) vascular cells,(13) and astro-
1Musculoskeletal Research Group, University of Manchester Medical School, Manchester, UK.
2Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK.
214

ESTROGEN RECEPTOR ␤ IN ADULT HUMAN BONE
215
cytes(14) are all positive for ER-␤. Furthermore, there is now 3-aminopropyltriethyoxysilane (Sigma Chemical Co.,
increasing evidence that tissues may express both receptors, Poole, UK).
either localized to different cell types(8,14) or to the same
cell.(15)
Preparation of polyclonal antibody to ER-␤
However, it is unclear how the two forms of the receptor
participate in the mechanism by which estrogen acts on
A peptide directed against the hinge (D) domain of hu-
bone. ER-␣ is expressed in human cells of the osteoblast man ER-␤(5) (CAGKAKRSGGHAPRVREL) was synthe-
lineage both in vitro(16,17) and in vivo.(18–21) There are sized by Affinity Reagents (Exeter, UK) and conjugated via
indications that this receptor is important to the action of the terminal cysteine residue to keyhole limpet hemocyanin
estrogen on the skeleton: epiphyses remain open and there is and injected into a sheep; immunizations and recovery of
marked bone loss in an adult man with a null mutation in antisera were all carried out by Diagnostics Scotland (Car-
ER-␣(22) and expression of the receptor protein is lost from luke, Lanarkshire, UK). Antibodies specific for ER-␤ were
osteoblasts and osteocytes in some cases of idiopathic os- affinity purified as described in detail in Saunders et al.(32)
teoporosis in men.(23,24) Nevertheless, although there is On Western blots the purified antibodies bound to recom-
some reduction in bone mineral density (BMD) of ER-␣ binant ER-␤ but not ER-␣, and immunoreactivity in control
knockout mice,(25) ovariectomy of these animals produced a tissues (rat epididymis) was abolished by incubation with
more profound bone loss,(26) implying that other receptors recombinant ER-␤ or immunising peptide, but not
may be important for estrogen action on the mouse skeleton. ER-␣.(32,33)
Indeed, there is evidence for ER-␤ expression in rat
bone.(27,28) However, skeletal abnormalities in ER-␤ knock-
out mice are surprisingly subtle in that the bone mineral
Immunocytochemistry
content of the cortices are increased only in adult females;
Sections were dewaxed in xylene, brought to absolute
males and immature animals remain unaffected.(29) The alcohol, and transferred to methanol containing 3% (vol/
question therefore remains as to the role of ER-␤ in estrogen vol) H2O2 for 30 minutes to block endogenous peroxidase.
action on the skeleton, especially in humans. It is expressed Antigen retrieval was then performed by placing slides in
in transformed fetal human cells of osteoblast lineage in 0.05 M glycine, containing 0.01% EDTA, pH 3.5, and
vitro(30) and in growth plate of adolescent boys and girls,(31) heating them for 5 minutes under full pressure in a stainless
but the exact cellular localization of ER-␤ in adult human steel pressure cooker (Tefal, Slough, Berkshire UK) and
bone is still unclear. Therefore, the aim of the present study leaving the slides in hot buffer for a another 20 minutes.
was to use a recently developed polyclonal antibody specific Sections were cooled, rinsed in Tris-buffered saline (TBS),
for ER-␤ to localize expression of the receptor protein by after which nonspecific binding was blocked with 20%
immunoperoxidase. Sections of bone were included from (vol/vol) normal rabbit serum (Dako, Ltd., Ely Cam-
adults with high bone turnover, to ensure the presence of a bridgeshire, UK) in 5% bovine serum albumin (BSA), made
variety of bone cells. We were thus able to show clear up in TBS, for 1 h at room temperature. They were then
nuclear localization of the receptor not only to cells of incubated with the antibody to ER-␤ diluted to 1:500 (for
osteoblast lineage but also in osteoclasts.
sections of benign hyperplasia of the prostate), 1:250, or
1:100 (for sections of bone/cartilage biopsy specimens) with
the rabbit serum and BSA in TBS. Stock concentrations of
the polyclonal antibody were at a concentration of 1.2 ␮g
protein/␮l. For some control experiments, the neat antibody
was either preadsorbed overnight at 4°C by a 10-fold
MATERIALS AND METHODS
Patients and biopsy specimens
Biopsy specimens from men with benign hyperplasia of protein concentration of the immunizing antigen or left in
the prostate were used as positive control tissue throughout contact with an equivalent volume of TBS, under iden-
the study. Bone biopsy specimens used in this investigation tical conditions. Both were then diluted to 1:500 for
were obtained from both men and women; fracture callus benign hyperplasia of the prostate or 1:250 or 1:100 for
was from patients between 21 and 74 years of age, as part bone, and sections remained in contact with antibody
of normal surgical procedures, pagetic bone and osteo- overnight at 4°C. In all experiments, control sections
phytes were from patients between 60 and 70 years of age, were included, which had been incubated with equivalent
and normal bone samples were from grafts used for maxil- concentrations of nonimmune sheep immunoglobulin G
lofacial repair after trauma in patients between 40 and 75 (IgG; Sigma Chemical Co.). Thereafter, sections were
years of age. Bone also was obtained from patients with incubated with biotinylated rabbit antibodies raised
hyperparathyroidism secondary to renal disease (50–70 against sheep IgG (Vector Laboratories, Peterborough,
years; n ϭ 2 for each type of biopsy). Approval had been UK), diluted 1:500 (vol/vol) with the rabbit serum and
given by the local Research Ethics Committee, and in- BSA in TBS, for 60 minutes at room temperature. Pri-
formed consent was obtained from the patients. All samples mary antibody binding to ER-␤ was then disclosed by
were fixed in 10% neutral buffered formalin, the bone standard avidin-biotin-peroxidase (Dako) followed by in-
biopsy specimens were demineralized in 20% EDTA until cubation with liquid diaminobenzidine (Sigma Chemical
radiologically decalcified (2–4 weeks), and then all biopsy Co.). Sections were counterstained with hematoxylin,
specimens were processed into paraffin wax. Sections (5 dehydrated, cleared, and mounted in XAM (BDH-Merk,
␮m) were cut and mounted onto slides coated with Poole Dorset, UK).

216
BRAIDMAN ET AL.
FIG. 1. ER-␤ expression in sections of benign hyperplasia of the prostate. Receptor was detected by immunoperoxidase, using the polyclonal
antibody, as described in the Materials and Methods section at a dilution of 1:500. (A) Immunoreactivity is localized to the nuclei of glandular
epithelial cells (solid arrows) and some hyperplastic cells. Some stromal cells also expressed ER-␤ (open arrows). When the antibodies were (B)
preadsorbed with immunizing peptide, immunoreactivity was blocked completely and sections appeared similar to (C) sheep IgG control (bar ϭ
66 ␮m).
ER-␤ completely blocked reaction product in all these cells
(Fig. 2F).
RESULTS
ER-␤ expression in benign hyperplasia of the prostate
Localization of ER-␤ in sections of benign hyperplasia of
the prostate is shown in Fig. 1. Retrieval of the receptor by
heating under pressure in acid conditions, as described in
ER-␤ expression in osteoclasts
These results are summarized in Fig. 3. Immunocyto-
the Materials and Methods section, was found to be essen- chemistry with the polyclonal antibody showed ER-␤ ex-
tial for its detection in sections of both the positive control pression in osteoclast nuclei in sections of fracture callus,
tissue and the bone. A primary antibody dilution of 1:500 osteophytes, pagetic bone, and normal bone. Most oste-
was optimal for benign hyperplasia of the prostate, which oclasts expressed the receptor, although in some cells, not
was used as a positive control throughout this study. Im- all the nuclei were positive (Fig. 3B). Little reaction product
munoreactivity was localized clearly to the nuclei of epi- was seen in the cytoplasm of the osteoclasts. Immunoreac-
thelial cells in hyperplastic glands, although there were far tivity was clearly visible in osteoclast nuclei even when the
fewer ER-␤ positive cells in the stroma. After preincubation polyclonal antibody was diluted to 1:500, in contrast to the
of antibody with immunizing peptide (Fig. 1B), sections other bone cells in which staining decreased when antibody
were devoid of reaction product and were similar to IgG was diluted more than 1:250 (data not shown). Osteoclast
controls (Fig. 1C).
ER-␤ expression was similar in bone from men and women.
Preincubation of the antibody with ER-␤ completely
blocked immunoreactivity in osteoclasts (Fig. 3E), which
was similar to IgG controls (Fig. 3F).
ER-␤ expression in intramembraneous and
endochondral bone
Osteoblasts in general were positive for ER-␤, and ex-
amples are shown of these cells from focal areas of in-
tramembraneous ossification in fracture callus (Figs. 2A and
2B) and in bone formation in osteophyte sections (Fig. 3C).
DISCUSSION
The study we present here shows that osteoclasts and
Some but not all osteocytes, especially within newly formed cells of osteoblast lineage clearly express ER-␤ protein.
bone, expressed ER-␤ (Fig. 2B). Immunoreactivity was That the polyclonal antibody used in the study was specific
localized to cell nuclei and was similar in bone from both for this isoform of ER is further confirmed by the results of
men and women. Immunoreactivity was not seen in hyper- preadsorption with antigen, recombinant protein, and by
trophic cartilage cells, within regions of endochondral os- Western blotting.(33) In our investigations and those of other
sification in either fracture callus (Figs. 2C and 3A) or researchers using fixed tissues(12,34,35) antigen retrieval by
osteophyte sections. In contrast, the adjacent mesenchymal heating was found to be essential for detection of ER-␤,
cells and some cells entrapped within the mineralized car- which was accomplished with relatively low concentrations
tilage were clearly ER-␤ positive, as were some of the cells of primary antibody, compared with those used in other
within small blood vessels adjacent to the cartilage (Fig. investigations, in which similar retrieval systems were not
2D). Megakaryocytes within the marrow (Fig. 2E) also used.(31,36) Although the use of heating, either under pres-
expressed the receptor. Preincubation of the antibody with sure or by microwave, can damage sections containing bone

ESTROGEN RECEPTOR ␤ IN ADULT HUMAN BONE
217
FIG. 2. ER-␤ expression in osteoblasts and chondrocytes. ER-␤ was localized by immunoperoxidase with a 1:250 dilution of the polyclonal
antibody, as described in the Materials and Methods section. (A) A region of intramembraneous ossification in fracture callus from a 28-year-old
man. Osteoblasts on the surface of the bone matrix (bm) are positive for the receptor; examples are shown by solid arrows, and immunoreactivity
is localized to nuclei. (B) Mesenchymal cells (mes) near active bone formation also are ER-␤ positive, as are some osteocytes within the bone
matrix, examples of which are shown by solid arrows (bar for A and B ϭ 32 ␮m). (C) An area of endochondral ossification from fracture callus.
Cells in the hypertrophic cartilage (hc) are negative, but those involved in endochondral bone formation (bf) or in the mesenchyme (mes), adjacent
to the initial stages of endochondral osteogenesis, are ER-␤ positive (bar ϭ 115 ␮m). (D) The inset is shown at higher magnification (bar ϭ 32
␮m). Hypertrophic cartilage cells within the cartilage are negative, but those at the perichondral edge and some of the cells in the small blood
vessel wall (solid arrow) are positive for receptor. (E) ER-␤ is localized to nuclei of megakaryocytes (solid arrows) in a patient with secondary
hyperparathyroidism. (F) Preadsorption of the polyclonal antibody with antigen blocks immunoreactivity from all cells in fracture callus; examples
of osteoblasts are shown by solid arrows (bar for E and F ϭ 32 ␮m).
and cartilage, we found that this was offset by the maximal mature human osteoclasts in vivo raises the possibility that
exposure of receptor epitopes.
estrogen may act on these cells directly.
Our results show that unequivocal expression of ER-␤ in
Osteoblasts were positive for ER-␤ in both endochondral
nuclei of osteoclasts of both men and women was positive and intramembraneous bone formation and in areas of remod-
for the receptor. The nuclear osteoclast receptor could be eling in adult bone. We and others have previously reported
detected with a lower concentration of antibody than in the ER-␣ expression in cells of osteoblast lineage,(19–21,45,46) al-
other bone cells, suggesting that osteoclasts have a relative though the studies from our laboratory indicated that only a
abundance of ER-␤. Although there are reports that estrogen proportion of osteoblasts in bone from adult women were
regulates osteoclast differentiation indirectly, through other positive for this receptor.(20) However, from our present results
cell types,(37) there also is evidence that the hormone can (Figs. 3A and 3B) ER-␤ expression appears to be widespread
affect mature osteoclasts directly.(38–41) Whether oste- in mature osteoblasts on bone surfaces. Some osteocytes also
oclasts express ER-␣ is controversial; some groups report were positive for the receptor, as we had found previously with
receptor expression either of messenger RNA (mRNA) or ER-␣.(18,20)
protein in mature cells,(19,39,42) but others show its expres-
We also observed that layers of mesenchymal cells, in
sion in immature osteoclasts but not in the mature healing fractures, or stromal cells, in other biopsy speci-
cells.(21,43,44) Furthermore, in a recent study of ER-␤ mRNA mens, were strongly positive for ER-␤ when adjacent to
expression, using in situ hybridization, expression of the sites of new bone formation, in agreement with a previous
receptor could not be localized equivocally to osteoclasts in report that receptor expression was localized to ossifying
neonatal rat bone,(28) although it is unclear if many suitable zones of fracture callus.(36) In this regard, it is noteworthy
cells were present in that study. However, osteoclasts, were that Arts et al.(30) found expression of ER-␤, rather than
readily visible in the biopsy specimens used in our investi- ER-␣, in cultured human fetal osteoblasts increased over the
gation and the clear expression of ER-␤ protein in nuclei of later stages of cellular differentiation up to mineralization.

218
BRAIDMAN ET AL.
FIG. 3. ER-␤ expression in osteoclasts. ER-␤ was localized by indirect immunoperoxidase as described in the Materials and Methods section.
(A) Osteoclasts, positive for ER-␤ (shown by solid arrows), are seen adjacent to mineralizing cartilage, undergoing endochondral ossification in
fracture callus from a 21-year-old woman. Note the absence of immunoreactivity in the hypertrophic chondrocytes (hc; bar ϭ 115 ␮m). (B) The
inset area is shown at higher magnification (bar ϭ 32 ␮m), in which ER-␤ expression is seen clearly in osteoclast nuclei, although there are some
that are negative (*). Note some mononuclear cells nearby also are positive for the receptor (open arrows). (C) Likewise, ER-␤ is expressed in
osteoclasts (shown by solid arrows) in sections of osteophyte from a man aged 67 years (bar ϭ 115 ␮m). (D) The inset is shown at higher
magnification (bar ϭ 32 ␮m). In panel C cells within the bone matrix (bm) and on bone forming surfaces (bf) are positive for the receptor. (E)
Immunoreactivity is blocked completely by preadsorbing the polyclonal antibody with the receptor; a facture callus section treated in this manner
is shown and (F) an IgG control is shown (bar ϭ 66 ␮m).
Whether the same cells express both receptors is a subject of mans(21) and animals(50) also express ER-␣. Overall, these
current investigation in our laboratory. If this were so, it results indicate that ER-␣ and ER-␤ are expressed either by
could provide an opportunity for heterodimerization of two the same or different cell types within sites of endochondral
isoforms(47,48) and, thereby, amplify the effects of estrogen ossification, which are therefore likely to be important tar-
on the bone cells. This might be especially important for gets for estrogen action.
men because their estrogen concentrations are low. Alter-
We also found ER-␤ protein expression in megakaryo-
natively, if ER-␣ and ER-␤ are expressed similarly in dif- cytes within bone marrow, in agreement with a preliminary
ferent cells of the osteoblast lineage, even low estrogen report from Khetawat et al.,(51) and in capillary blood ves-
concentrations could influence a wide spectrum of gene sels. Lindner and colleagues have described ER-␤ mRNA
expression and thereby a variety of biological effects. A expression in the aortic endothelium and smooth muscle of
similar situation has been discussed for neural tissue(49) and male rats, both in vivo and in vitro,(13) although Baker et al.
might support a wide distribution of ER-␤ in the skeleton. were unable to detect mRNA or protein for either ER-␣ or
Distribution of ER-␤ expression in cartilage appeared to ER-␤ in human umbilical veins.(52) The presence of ER-␤ in
be localized specifically. Hypertrophic chondrocytes, in small capillary vessels and in the megakaryocytes implies
fracture callus, were largely negative, although cells within that they may respond to estrogen, which thus may be able
centers of endochondral bone formation were positive for to exert additional influences on bone through these cells.
the receptor. However, in growth plate from adolescent
Overall, in the cells that were positive for ER-␤, we found
girls, hypertrophic chondrocytes are reported to express that immunoreactivity was expressed in nuclei. Although
ER-␤ but proliferative and resting cartilage cells are nega- others have reported expression of this receptor in oste-
tive.(31) Whether hypertrophic chondrocytes have different oclasts,(36) they found that it was localized to the cytoplasm
ER-␤ expression when in growth plate compared with frac- and not nuclei of these cells. It is uncertain whether this is
ture callus, or whether the different antibodies used in the because of differences in immunoperoxidase technique or of
two studies influence detection of this receptor, is uncertain. the type of biopsy specimens studied. Our investigation
Nevertheless, growth plate chondrocytes in both young hu- focused on bone from adults, in which we found no obvious

ESTROGEN RECEPTOR ␤ IN ADULT HUMAN BONE
219
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ER-␤ expression may be an important feature of its regu-
lation in bone.
ACKNOWLEDGMENTS
We thank Action Research for supporting this study
(project grant A/P/0727).
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Address reprint requests to:
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Musculoskeletal Research Group
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cursor cells of neonatal rats by fluorescence in situ hybridiza- Received in original form March 28, 2000; in revised form August
tion. J Mol Endocrinol 20:211–219. 9, 2000; accepted October 6, 2000.