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1 Academic Rheumatology and 2 Orthopaedic Surgery Units, University of Bristol, Bristol BS2 8HW, United Kingdom; and 3 Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We recently found that
estrogen receptor (ER) antagonists prevent high-dose estrogen from
inducing the formation of new cancellous bone within the medullary
cavity of mouse long bones. In the present investigation, we studied
the role of specific ER subtypes in this response by examining whether
this is impaired in female ER
/ mice previously
generated by targeted gene deletion. Vehicle or 17-estradiol
(E2) (range 4-4,000
µg · kg1 · day1) was
administered to intact female ER/ mice and wild-type
littermates by subcutaneous injection for 28 days. The osteogenic
response was subsequently assessed by histomorphometry performed on
longitudinal and cross sections of the tibia. E2 was found
to cause an equivalent increase in cancellous bone formation in
ER/ mice and littermate controls, as assessed at the
proximal and distal regions of the proximal tibial metaphysis.
E2 also resulted in a similar increase in endosteal mineral
apposition rate in these two genotypes, as assessed at the tibial
diaphysis. In contrast, cortical area in ER/ mice
was found to be greater than that in wild types irrespective of
E2 treatment, as was tibial bone mineral density as
measured by dual-energy X-ray absorptiometry, consistent with previous reports of increased cortical bone mass in these animals. We conclude that, although ER acts as a negative modulator of cortical modeling, this isoform does not appear to contribute to high-dose estrogen's ability to induce new cancellous bone formation in mouse long bones.
osteoblasts; estrogen receptor; histomorphometry
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ESTROGEN EXERTS an important protective effect on the skeleton, as illustrated by the significant bone loss associated with estrogen deficiency (17), which is prevented by hormone replacement (8, 24). This ability of estrogen to prevent bone loss is thought to reflect two distinct actions. First, numerous clinical and animal studies indicate that estrogen acts to suppress osteoclastic bone resorption, leading to a decrease in bone turnover (7, 23, 32). In addition, estrogen has been reported to stimulate osteoblast function when administered at a relatively high dose, as assessed in studies of postmenopausal women receiving estradiol implants (9, 26) and in rodent models (2, 6). Although higher doses of estrogen are associated with extraskeletal effects that may limit their use in postmenopausal women, improved understanding of the molecular basis for estrogen's actions on bone may provide the basis for developing novel therapeutic agents capable of targeting these.
To explore the basis for estrogen's stimulatory action on osteoblasts
in more detail, we exploited previous observations that high-dose
estrogen induces an exaggerated osteogenic response in mouse long bones
(3, 25). In time course studies, we found that this
response consists of the generation of new cancellous bone formation
surfaces, associated with a marked expansion in the bone marrow content
of early osteoblast precursors (13, 15, 20). Despite the
fact that high doses of estrogen are required to induce a maximal
osteogenic response in this species, we also observed that this can be
inhibited by an estrogen receptor (ER) antagonist, suggesting that an
ER-dependent mechanism is involved (19). Since the recent
cloning of the -isoform of the ER from a rat prostate cDNA library
(11), it is now recognized that the ER exists in at least
two distinct isoforms. In view of observations that ER is expressed
at relatively high levels in osteoblasts as assessed both in vitro and
in vivo (1, 4, 5, 12, 27, 30), it is possible that this
isoform contributes to estrogen's stimulatory action on osteoblasts in
mice as described above.
The generation of mice with a targeted deletion in the ER gene
(ER/ mice) has provided an opportunity to
explore the role of ER in regulating skeletal responses to estrogen.
On the basis of observations that cortical bone mass is increased in
female animals, ER has been suggested to act as a negative modulator
of cortical bone growth (31). Although trabecular bone
density in young adult female ER/ mice is unaffected
(31), cancellous bone volume appears to be increased in
older animals, associated with enhanced osteoblast activity
(29), suggesting that ER also suppresses osteoblast activity in cancellous bone. In the present investigation, we aimed to
explore the role of ER in regulating osteoblast activity in female
mice by determining whether this isoform contributes to the stimulatory
action of high-dose estrogen on cancellous bone formation, as assessed
in long bones of female ER/ animals.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental design.
ER/ mice were generated at the University of North
Carolina, back-crossed onto a C57Bl/6 genetic background, transferred
to the University of Bristol animal facility, and subsequently crossed with wild-type C57Bl/6 mice from the local breeding stock
(10). PCR-based genotyping was performed on DNA extracted
from tail tips at 4-6 wk of age on the basis of previously
published primer sets. Intact 12-wk-old female ER/
mice and age-matched wild-type littermates were subsequently administered vehicle [0.1 ml corn oil (Sigma, Poole, Dorset, UK)] or
4, 40, 400 or 4,000 µg/kg 17-estradiol (E2; Sigma) by
daily subcutaneous injection (4-6 animals/group). This protocol
was employed on the basis of our previous study (19),
where we defined the dose responsiveness of estrogen-induced
osteogenesis in wild-type intact female mice.
Histomorphometry. All histomorphometry was performed at the proximal tibial metaphysis and tibial diaphysis. Tibiae were cleared of soft tissue, separated into proximal and distal halves, fixed in 70% ethanol for 48 h, and then dehydrated through a graded series of alcohols: 80% ethanol, 90% ethanol, and then three changes of 100% ethanol for 24 h each. Tibiae were then cleared in chloroform for 24 h, placed for a further 24 h in 100% ethanol, and embedded without decalcification in LR White Hard Grade (London Resin, Reading, UK). Longitudinal sections of the proximal portion of the tibia were then prepared for histomorphometric analysis of the proximal tibial metaphysis by use of a Reichert-Jung 2050 microtome with a "d" profile tungsten carbide knife. Sections (7 µm) were stained with 1% toluidine blue in 0.01 M citrate phosphate buffer for bone area measurement, and 10-µm sections were mounted unstained in Fluoromount (BDH; Laboratory Supplies, Poole, UK) for assessment by fluorescent microscopy. For analysis of the tibial diaphysis, 15-µm cross sections of the distal tibial portion were obtained immediately proximal to the tibiofibular junction and prepared as above.
Histomorphometric analysis was performed using transmitted and epifluorescent microscopy linked to a computer-assisted image analyzer (Osteomeasure; Osteometrics, Atlanta, GA). Two nonconsecutive sections per animal were analyzed for each parameter in a blinded manner. For the proximal tibial metaphysis, two sampling sites, each with a standard area of 0.36 mm2, were analyzed as previously described (20). The proximal border of the proximal sampling site was situated 0.25 mm below the growth plate to exclude primary spongiosa (area 1); the second sampling site was immediately distal to the first sampling site (area 2). Cancellous bone area was expressed as a percentage of total tissue area [bone area (BV)/tissue area (TV)]. The length of trabecular bone perimeter covered by double label (dlS) was expressed with reference to the total tissue area (tissue area referent: dlS/TV) and as a percentage of the total length of cancellous bone perimeter (BS; cancellous perimeter referent: dlS/BS). The former parameter (i.e., dlS/TV) was analyzed, because this gives a better reflection of estrogen's tendency to induce the appearance of new sites of cancellous bone formation than dlS/BS does (20). Mineral apposition rate (MAR) was determined by dividing the mean distance between the tetracycline and calcein labels by the time interval between the administration of the two labels (values were not corrected for the obliquity of the plane of section). Cortical bone parameters were assessed on cross sections of the tibial diaphysis. Cross-sectional and medullary area were analyzed on toluidine blue-stained sections, and cortical area was derived from their difference. Periosteal and endocortical dlS/BS were assesssed on unstained sections by measuring the proportion of, respectively, periosteal and endocortical surface covered by double fluorochrome label. Periosteal and endocortical MAR were derived from interlabel distance at these two surfaces.Statistical analysis.
Results are expressed as means ± SE. An unpaired Student's
t-test was used to examine baseline differences between
wild-type and ER/ mice. Two-way analysis of variance
was subsequently performed to examine whether E2 dose or
genotype exerted a statistically significant effect on the measured
parameters and to study possible interactions between these two
variables. The cut-off for statistical significance was taken as
P = 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Histological assessment suggested that E2 induced the
formation of new cancellous bone in the proximal tibial metaphysis to an equivalent extent in wild-type and ER/ intact
female mice (Fig. 1). This finding was
confirmed by subsequent histomorphometric analysis, which revealed that
E2 caused a similar increase in dlS/TV and BV/TV to that
previously observed (19) in both ER/
mice and wild-type littermates (Fig. 2).
These two genotypes also showed equivalent changes in dlS/TV and BV/TV
at the more distal metaphysis region (Fig.
3). Although E2 had less
tendency to stimulate MAR, our results were suggestive of a small
increase at the dose of 400 µg/kg to a similar extent in
ER/ mice and littermate controls (Table
1). In contrast to the response in dlS/TV, E2 did not increase dlS/BS, and, if
anything, maximal doses E2 tended to suppress this
parameter in both ER/ and control animals.
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On the basis of previous evidence that estrogen stimulates bone
formation at the endocortical surface of mouse long bones, we further
compared the osteogenic response of ER/ and
wild-type mice by analyzing parameters of endocortical bone formation.
E2 caused a similar increase in endocortical MAR in both
genotypes (Fig. 4). In contrast,
endocortical dlS/BS showed no significant response to E2
and was reduced in ER/ mice compared with wild-type
animals. E2 caused a significant suppression in periosteal
dLS/BS (Fig. 5), in keeping with
estrogen's inhibitory action on periosteal bone growth. However, this
suppression appeared to be less marked in ER/ mice,
as illustrated by the significant interaction between estrogen dose and
genotype.
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Cortical area and cross-sectional area were both significantly
increased in ER/ mice compared with littermate
controls (Table 2). Tibial BMD was also
significantly higher in ER/ mice, but, unlike
cortical area and cross-sectional area, this parameter additionally
showed an increase after E2 treatment (Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We compared the osteogenic response of wild-type and
ER/ intact female mice with exogenous E2
by use of the same experimental protocol as that used to define the
dose-response profile of this action in wild-type animals
(19). E2 was found to cause a similar increase
in the extent of cancellous mineralizing surfaces to that previously
observed in both wild-type and ER/ mice. Because
estrogen-induced osteogenesis progresses from proximal to distal within
the proximal tibial metaphysis (20), partial suppression
may be best detected by analyzing more distal regions of interest
(18, 21). However, in the present study, a similar estrogenic response was seen in the two genotypes even when the distal
region was examined. Estrogen-induced osteogenesis in mouse long bones
has also been reported to involve the endocortical surface
(2). Consistent with this finding, we noted an increase in
endocortical MAR after E2 administration that was not
significantly different between the two genotypes. These findings
indicate that intact female ER/ mice show an
equivalent osteogenic response to E2 to that observed in
wild-type animals.
We (19) previously found that E2 doses, as
administered in the present study, result in serum E2
levels within the upper physiological range and beyond. Although
E2 levels were not measured in the present study,
equivalent changes are likely to have occurred, in light of previous
evidence that serum E2 levels are similar in wild-type and
ER/ mice (29). Taken together, these
findings suggest that ER is not necessary for the osteogenic action
of high estrogen levels in intact female mice. Presumably, a different
ER isoform plays a major role in this response, such as ER
. Our
preliminary findings from studies of ER/ mice are
consistent with this suggestion.
Our observations raise the possibility that, in addition, ER is not
required for the osteogenic action of lower levels of estrogen within
the physiological range. The finding in the present study that indexes
of cancellous bone formation are equivalent in untreated
ER/ and wild-type animals, and previous reports that
trabecular BMD is similar in these two genotypes (31), are
consistent with this possibility. Previous observations that
ER/ mice are partially protected against age-related
bone loss suggest that, if anything, ER acts as a negative regulator
with respect to effects of physiological estrogen levels on bone
formation, which may reflect inhibition by ER of ER expression or
ER-dependent transcription (14, 29). However, there is
no indication from the present study that the osteogenic response to
high-dose E2 is enhanced in female ER/ mice.
Cross-sectional area and cortical area of the tibial diaphysis were
significantly higher in ER/ mice, which is
presumably the explanation for our finding that tibial BMD in these
animals was also elevated. Because similar differences in diaphysial
areas and tibial BMD were observed between genotypes irrespective of
E2 dose, they are likely to reflect differences in the
pretreatment baseline rather than any altered responsivess to estrogen.
The suggestion that cortical bone mass is increased in female
ER/ mice irrespective of estrogen treatment is in
line with previous phenotypic studies by Windahl et al.
(31). A likely explanation for this observation is that
ER contributes to estrogen's inhibitory action on bone formation at
the periosteal envelope. The finding in the present study that
estrogen-induced suppression of periosteal dlS/BS is reduced in
ER/ female mice is consistent with this possibility.
Taken together, our observations suggest that, whereas ER plays a
significant role in mediating the inhibitory effects of physiological
estrogen levels on cortical modeling, this isoform is not required for
the osteogenic response of cancellous bone of intact female mice to
high-dose estrogen. Although ER is expressed by trabecular
osteoblasts and stromal cells, as assessed in neonatal human and rat
bone (4, 30) and adult human bone (5),
presumably, these do not represent the effector cell population that
mediates estrogen-induced osteogenesis in female mice. Estrogen exerts other effects, which were not examined in the present study, in cancellous bone in which ER might play an important role, such as
inhibition of osteoclastic bone resorption and suppression of
hematopoiesis (3, 13). After recent findings that
estrogen-induced osteogenesis in female mice involves stromal cells and
osteoblast precursors that express bone morphogenetic protein (BMP)-6
and the core binding transcription factor Cbfa1, respectively
(15, 16), further studies are planned to determine whether
these cells preferentially co-express ER or ER.
In view of our findings that suggest that ER is not necessary for
estrogen-induced osteogenesis in female mice, it is tempting to
speculate that this isoform plays little role in estrogen's stimulatory action on osteoblasts in postmenopausal women. One implication of this conclusion is that novel ER-selective ligands, which have recently been developed (28), may not confer
significant advantages in the treatment of postmenopausal osteoporosis
compared with conventional approaches. However, any extrapolation from mice to humans should be treated with caution in view of significant species differences with respect to the skeletal actions of estrogen. For example, rather than inducing the appearance of new sites of
cancellous bone formation, in humans, estrogen predominantly acts to
increase mean wall thickness (9, 26). To what extent these
responses in different species are functionally related, for example
due to common effects of estrogen on osteoblast precursors, is
currently unclear.
In a recent study based on a different line of ER/
mice, young adult female knockout mice were found to have normal
cortical bone mass but increased cancellous bone volume and reduced
bone resorption (22). These observations, which suggest
that different lines of ER/ mice have distinct
skeletal phenotypes, were thought to reflect initial reports that
ER/ mice as used in the present study express a
mutant form of ER that can bind estradiol but is unable to activate
gene transcription (10). However, after reanalysis of
variant ER transcript sequences in ER/ mice
utilized in this investigation, these were uniformly found to have stop
codons (J.B. Hodgin and O. Smithies, unpublished observations),
and so there is no possibility of any mutant ER protein being
expressed. Nevertheless, although the reasons for the apparent
difference in skeletal phenotype of these two lines of
ER/ mice are unclear, it may be informative to
repeat our investigations with the use of ER/ mice
generated by other targeting strategies.
In conclusion, we have found that estrogen-induced osteogenesis in
intact female mice is unaffected by targeted gene deletion of ER.
Therefore, although ER has previously been suggested to act as a
negative modulator of cortical bone modeling in mice and is reported to
be highly expressed in the metaphysis, it does not appear to be
necessary for the stimulatory effect of high-dose estrogen on
cancellous bone formation. Further studies are required to determine
whether the actions of high levels of estrogen on osteoblasts in other
species such as humans are likewise independent of ER.
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ACKNOWLEDGEMENTS |
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This work was supported by the Nuffield Foundation (Oliver Bird Fund RHE/00031/G) and National Institutes of Health (GM-20069).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. H. Tobias, Rheumatology Unit, Bristol Royal Infirmary, Bristol BS2 8HW, UK (E-mail: Jon.Tobias{at}bristol.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00071.2002
Received 19 February 2002; accepted in final form 30 May 2002.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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) and ER
) mice. Animals (4-7/group) were administered
E2 0, 4, 40, 400, or 4,000 µg/kg by daily sc injection
for 28 days. A: double-labeled perimeter/tissue area
(dlS/TV). B: bone area/tissue area (BV/TV). Results show
means ± SE. Two-way ANOVA revealed a significant effect of dose
(P < 0.0001).
