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Original Studies |
and ß Are Differentially Expressed in Developing Human Bone1
Address all correspondence and requests for reprints to: Dr. Sharyn Bord, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Box 157, Cambridge, United Kingdom CB2 2QQ. E-mail: sb201{at}cam.ac.uk
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Abstract |
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and ß. The aim of this study was
to establish the cellular distribution of ER and ERß in neonatal
human rib bone. ER and ERß immunoreactivity was seen in
proliferative and prehypertrophic chondrocytes in the growth plate,
with lower levels of expression in the late hypertrophic zone.
Different patterns of expression of the two ERs were seen in bone. In
cortical bone, intense staining for ER was observed in osteoblasts
and osteocytes adjacent to the periosteal-forming surface and in
osteoclasts on the opposing resorbing surface. In cancellous bone,
ERß was strongly expressed in both osteoblasts and osteocytes,
whereas only low expression of ER was seen in these areas. Nuclear
and cytoplasmic staining for ERß was apparent in osteoclasts. These
observations demonstrate distinct patterns of expression for the two ER
subtypes in developing human bone and indicate functions in both the
growth plate and mineralized bone. In the latter, ER is
predominantly expressed in cortical bone, whereas ERß shows higher
levels of expression in cancellous bone. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ER, a nuclear hormone receptor, is a member of a family of
activated transcription factors that can initiate or enhance the
transcription of genes containing specific hormone response elements.
The human ER protein consists of six functional domains
(5) with two regions involved in transcriptional activity.
Two ERs have been identified, and ß, which are distinct proteins
encoded by separate genes located on different chromosomes. ER
consists of 595 amino acid residues with a molecular mass of 66 kDa
(6); the more recently cloned ERß consists of 485 amino
acid residues with a molecular mass of 54 kDa (7). They
exhibit considerable homology in the DNA- and ligand-binding domains
and have similar relative binding affinities. However, the N-
terminal regions of ER and ERß are poorly conserved, with only
20% amino acid homology.
Several studies have demonstrated the presence of ER messenger
ribonucleic acid (mRNA) in osteoblasts and osteoblast-like cell lines.
Arts et al. (8) showed differential mRNA
expression of ER and -ß during osteoblast differentiation, with
ERß increasing gradually throughout culture while ER levels
remained constant after day 10. Braidman et al.
(9) reported ER mRNA expression in human osteoblasts
and ER protein in osteocytes, and Lim et al.
(10) demonstrated mRNA for both subtypes in cancellous
bone, but not cortical bone, with expression being lost after
ovariectomy. In separate experiments Windahl et al.
(11) and Onoe et al. (12)
described the presence of ERß mRNA in rat bone, whereas Vidal
et al. (13) demonstrated ERß immunoreactivity
in human callus tissue from healing fractures. ERß has been
immunolocalized in human growth plate cartilage (14);
however, the coexpression of ER and ERß protein in bone has not
been reported. Thus, the aim of this study was to examine the
expression of ER and ERß in developing human bone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histology and immunolocalization
For histology, sections were stained with Diff-Quick (Baxter Dade AG) or hematoxylin and eosin. Immunolocalization was carried out on both frozen and wax-embedded sections using an indirect immunoperoxidase system as previously described (15). Some sections were lightly counterstained with Gill’s hematoxylin (1:100, 20 s) to provide morphological detail. Sections were mounted in Vectamount (Vector Laboratories, Inc., Peterborough, UK) and observed under brightfield microscopy on a Nikon E-800 microscope (Kingston, UK).
Primary antibodies to ER (0.5 µg/mL rabbit polyclonal HC-20,
mapping to the carboxyl-terminus of the ER and shown not to
cross-react with ERß) and ERß (0.2 µg/mL goat polyclonal L-20,
mapping to the carboxyl-terminus of the ERß and shown to be
noncross-reactive with ER) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit polyclonal
antibody raised against the N-terminal amino acid residues 55–70 of
ERß (2.0 µg/mL; PAI-311, Affinity BioReagents, Inc., Golden, CO) was also used. Biotinylated antirabbit and
antigoat (Vector Laboratories, Inc.) were used as
secondary antibodies. All antibodies were diluted in 0.1% BSA
(Vector Laboratories, Inc.) in PBS. Rabbit (2.0 µg/mL)
and goat (1.0 µg/mL) IgG and omission of primary antibodies were used
as controls. In addition, antibodies were treated with specific
immunizing peptides (10-fold excess for 2 h at room temperature)
and then applied as previously described for primary antibodies.
Paraffin wax-embedded samples of endometrium and colon were used as
positive and negative control tissues to confirm the specificity of ER
antibodies.
Western blotting
The specificity of antibodies was also confirmed by Western
blotting. Osteoblastic cells were grown to confluence in T75 flasks
with the media and conditions detailed below. Cells were removed with
TRIzol and protein extracted according to the manufacturer’s
instructions (Life Technologies, Inc., Paisley, UK).
Protein extracts were separated on 10% SDS-PAGE under reducing
conditions and transferred to Hybond polyvinylidene difluoride membrane
(Amersham Pharmacia Biotech, Aylesbury, UK). After
blocking overnight, the membranes were incubated with primary
antibodies (ER, 1 µg/mL; ERß, 2 µg/mL), and protein-antibody
complexes were visualized using horseradish peroxidase-conjugated
secondary antibodies. The signal was enhanced with chemiluminescence
(ECL kit, Amersham Pharmacia Biotech). Molecular weight
standards were run on all gels.
Cell culture
A primary human osteoblastic cell line (CLONTECH Laboratories, Inc., Basingstoke, UK) was grown to confluence and seeded in eight-well chamber slides (Labtech International, Ringmer, UK) at 104 cells/mL in McCoy’s 5A supplemented with 10% FBS (Life Technologies, Inc.), penicillin/streptomycin/glutamate (Life Technologies, Inc.), and ascorbic acid (100 µmol/L; Wako, Alpha Labs, Eastleigh, UK). After 24-h incubation at 37 C in a humidified chamber with 5% CO2, the medium was removed and replaced with fresh medium for an additional 48 h. The medium was removed, and the cells were rinsed with PBS and then fixed in 4% paraformaldehyde for 5 min at room temperature. After extensive washing with PBS, immunolocalization was performed as detailed above. Positively staining cells were counted in five randomly selected fields of view and expressed as a percentage of the total cell count.
Primary human osteoclast-enriched cultures were obtained from
peripheral blood. Monocytes were prepared by layering freshly collected
human blood over equal volumes of Histopaque 1077 (Sigma)
at room temperature and were centrifuged at 400 x g
for 30 min at 4 C. The monocyte cell-rich interface layers, which
appear as an opaque band, were collected, added to PBS (Mg and Ca free)
to a total volume of 45 mL, and centrifuged (250 x g,
10 min, 4 C) to remove any remaining platelets. The supernatant was
removed, and the cellular pellet was resuspended in 20 mL PBS and
centrifuged. Collected cells were resuspended in 10 mL MEM
(Life Technologies, Inc.) and plated at 1.5 x
106 cells/mL on dentine slices in 24-well plates
in medium supplemented with 10% FBS, penicillin/streptomycin/glutamate
(Life Technologies, Inc.), receptor activator of nuclear
factor-
B ligand (25 ng/mL; Insight Biotechnology, Wembley, UK), and
macrophage-colony stimulating factor (25 ng/mL; R & D Systems, Abingdon, UK). After overnight culture the plates were
gently rocked to remove nonadherent cells, and the medium was replaced.
Thereafter, the medium was changed every 2–3 days for 3 weeks. At the
end of the culture period, the medium was removed, and cells were
washed briefly with PBS and fixed in 4% paraformaldehyde for 5 min at
room temperature. After extensive washing in PBS, the cells on the
dentine slices were immunolocalized as detailed above. Confirmation of
osteoclast phenotype was determined by observation of resorption pits,
tartrate-resistant alkaline phosphate staining, and calcitonin
receptor positivity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß were highly and
differentially expressed in samples of endometrium, whereas in samples
of colon only ERß demonstrated nuclear staining in cells of the
mucosa. Sections treated with preimmune serum of matched IgG
concentrations to primary antibodies, omission of primary antibody, and
antibody incubated with the immunizing peptide showed absence of
staining. Western blotting showed distinct bands at 66 and 54 kDa
corresponding to ER and ERß, respectively (Fig. 1
). The two ERß antibodies used
exhibited similar staining patterns.
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Human osteoblast-like cells cultured for 48 h showed intense
nuclear ER immunoreactivity in many cells (Fig. 2A). ERß was also highly expressed in
osteoblasts, with expression localized in the nuclear regions (Fig. 2B). Generally, ER was expressed in a greater number of cells than
ERß (46% and 35%, respectively, of cells staining positively).
Cells incubated with rabbit or goat IgG at the same concentrations as
the primary antibodies showed an absence of staining (Fig.
2C).
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and
ERß, demonstrating both nuclear and cytoplasmic staining (Fig. 2
, D
and E). Surrounding mononuclear cells, possibly osteoclast precursors,
also showed ER and, to a lesser extent, ERß positivity. Cells
localized with rabbit or goat IgG showed an absence of staining
(Fig. 2F). Histology
Longitudinal sections of rib bone stained with Diff-Quik or
hematoxylin-eosin showed resting chondrocytes that progressed into
columns of proliferating chondrocytes as endochondral ossification
proceeded. Chondrocytes underwent progressive hypertrophy with
increasing mineralization of the intercolumnar matrix. Within the
primary spongiosa, osteoid was deposited on remnants of resorbed
cartilage to form spicules of bone, which were then remodeled by
osteo-clasts and osteoblasts (Fig. 3, A and B). A surrounding periosteal collar of mineralized woven bone,
containing many osteocytes, was covered by periosteum and contained
vascular channels.
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No age- or gender-related differences in expression were observed
between the samples, and all samples exhibited similar staining
patterns. Sections immunolocalized with preimmune serum showed no
staining (Fig. 3C). Comparison of cryosections and wax-embedded
sections revealed similar staining patterns in all bone areas, although
there were differences in cartilage. In cryosections, both ER and
ERß were expressed by chondrocytes within the growth plate, with the
most intense staining in early hypertrophic cells. Decreased expression
was evident in the late hypertrophic and mineralizing zones.
Chondrocytes at the chondro-osseous junction were strongly
immunoreactive for ER and ERß. On wax-embedded sections the
expression was greatly decreased. Most samples demonstrated only weak
signal for both ER and ERß in the early hypertrophic zone and in
chondrocytes adjacent to the chondro-osseous junction, with no staining
in other areas.
Within the bone a greater degree of differential expression was seen,
with ER being more evident in cortical bone and ERß being the
dominant form in cancellous bone. Osteocytes in the cortical bone
collar demonstrated intense ER immunoreactivity, with the majority
of cells staining positively. Osteoblasts within osteons and on the
periosteal and endosteal surfaces were also strongly positive for ER
(Fig. 3D). In contrast, within the cancellous bone, only low levels of
ER expression were observed in osteocytes, usually most pronounced
in cells within and close to the newly mineralizing osteoid (Fig. 3E).
Low level ER staining was seen in osteoblasts at cancellous sites
(Fig. 3F). Many osteoclasts showed positive nuclear and cytoplasmic
ER immunoreactivity (Fig. 3G), particularly those on the periosteal
surface of the cortical collar.
The pattern of staining for ERß differed significantly to that of
ER. In cortical bone most osteocytes showed low or no ERß
immunoreactivity. Osteoblasts at these sites generally showed little
ERß activity, but some staining was detected in osteoblasts on the
endosteal surface (Fig. 3H). In contrast, in cancellous bone osteocytes
demonstrated strong ERß immunoreactivity (Fig. 3I), and intense
staining was seen in osteoblasts apposed to bone spicules within the
primary spongiosa and on bone surfaces in the more mature cancellous
areas (Fig. 3J). Many osteoclasts stained positively for ERß (Fig. 3K), but this was most pronounced in osteoclasts in the primary
spongiosa.
ER and ERß were detected in cells within marrow spaces and
exhibited different patterns of staining along the length of the rib.
ERß was most highly expressed in the primary spongiosa marrow space
cells, with decreasing intensity of staining in marrow cells toward the
mature cancellous bone. In contrast, ER immunoreactivity was most
evident in marrow cells within mature cancellous bone, with decreasing
expression in marrow cells approaching the primary spongiosa.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß in developing human bone. Their differential
expression in the growth plate and mineralized bone, with ER more
highly expressed in cortical than in cancellous bone and ERß most
evident at cancellous than cortical sites, suggests that they may have
different functions. In addition, we have demonstrated in
vitro the presence of ER and ERß in cultured human
osteoblasts and human osteoclasts.
Our observations of the distribution of the ER subtypes in bone are
consistent with the findings of Onoe et al.
(12) of ERß mRNA in cancellous bone of 8-week-old rats;
these researchers speculated that as ovariectomy induces bone loss
preferentially in cancellous bone, estrogen may predominantly regulate
cancellous bone remodeling. Similarly, the study by Lim et
al. (10) reported the presence of ER and ERß
mRNA in cancellous rat bone, with expression being lost after
ovariectomy, but they were unable to detect either subtype in cortical
bone before or after ovariectomy. In contrast, Ehrlich et
al. (16) reported the presence of ER in many newly
incorporated cortical bone osteocytes, but with lower expression in
osteocytes in the cortical mineralized matrix. Our experimental design
did not allow direct comparison of the relative levels of expression of
the two subtypes, and further studies are required to assess the
functional implications of their differential distribution in cortical
and cancellous bone. One possibility is that in human neonatal bone,
estrogen-dependent regulation of cancellous bone remodeling may be
predominately mediated by ERß, whereas cortical bone regulation
occurs mainly through ER. Alternatively, in view of the recent
evidence that ERß can inhibit ER activity (17), the
differential distribution of the subtypes may indicate site-specific
variations in estrogen responsiveness within the skeleton.
The exact mechanisms by which the skeletal effects of estrogen are
mediated are still unknown, but the importance of ERs in human bone
metabolism is supported by the report of severely retarded skeletal
maturation in a man with estrogen resistance due to a mutation in the
ER gene (18). Studies using mice deficient in one or
another of the ERs have produced inconclusive results, but suggest that
age and gender are influential. Mice lacking functional ER were
found to have only minor skeletal abnormalities (19),
suggesting that ER is not a major regulator of bone development in
this species (20). In another study female ER null mice
were found to have decreased femur length and diameter and a slight
reduction in femur bone mineral density and bone mineral content,
whereas males had a more marked reduction in bone mineral density. In
the ERß knockout mice it has been reported that adult females exhibit
changes in cortical, but not cancellous, bone, whereas prepubertal and
male mice are unaffected (21). Based on the results of
single and double receptor knockout experiments, Windahl et
al. (22) suggested that ERß was involved in both
the regulation and maintenance of cancellous bone in female mice,
whereas Sims et al. (23) postulated that ER
was required for normal cortical bone growth in males, although in
females ERß could compensate for the loss of ER. However, as
discussed above, the reported ability of ERß to antagonize ER
activity adds complexity to the interpretation of these experiments,
and the functions of the two subtypes remain to be clearly defined.
There have been conflicting reports of ER and ERß in growth plate
chondrocytes. Nilsson et al. (14, 24)
identified immunoreactivity for both receptors only in the hypertrophic
zone; similar findings were reported by Batra et al.
(25), who identified ERß exclusively in hypertrophic
cells in wax- embedded sections from human fracture callus. Kusec
et al. (26) showed ER expression in resting,
proliferative, and hypertrophic zones, and Connor et al.
(27) demonstrated ER and ERß mRNA throughout all
regions in frozen sections of cartilage. In our study we investigated
both frozen and paraffin wax-embedded sections and found
immunoreactivity for both ERs in all regions in cryosections, with the
highest levels of expression seen in the early hypertrophic zone and in
chondrocytes adjacent to the chondro-osseous junction. The expression
seen in paraffin-embedded sections was much weaker, with signal in some
samples only detected in hypertrophic chondrocytes, indicating that in
these sections, antigenicity may be masked or stripped by
decalcification. Thus, features related to methodology may explain at
least in part the conflicting results reported in previous studies.
Our results demonstrate the presence of both ER and ERß in
osteoclasts. Previously, ER mRNA and protein binding have been
reported in avian osteoclasts (28), and ER
immunostaining has been seen in isolated human osteoclasts
(29), whereas ERß mRNA has been detected in human fetal
osteoclasts (27). Recently, Hainey et al.
(30) reported nuclear ERß expression in human
osteoclasts in vivo. Our study extends these observations by
providing in vitro and in vivo evidence for both
receptors in human osteoclasts.
In summary, this study provides evidence for the presence of ER and
ERß in developing human bone. The differential expression of the two
receptors suggests that they may have different functions, particularly
with respect to cortical and cancellous bone.
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Acknowledgments |
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Footnotes |
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Received October 17, 2000.
Revised January 23, 2001.
Accepted February 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß during differentiation of
human osteoblast SV-HFO cells. Endocrinology. 138:5067–5070. and ß mRNA abundance in rats and the effect of
ovariectomy. J Bone Miner Res. 14:1189–1196.[CrossRef][Medline]
transcriptional activity and is a key regulator of the cellular
response to estrogens and antiestrogens. Endocrinology. 140:5566–5578., ß and double
knockouts reveals a dual role for ERß in bone remodelling. J
Bone Miner Res. 15:S160.
mRNA. Bone.
23:F049.
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