Estrogen-induced osteogenesis in intact female mice lacking ERbeta

K. E. McDougall1, M. J. Perry2, R. L. Gibson1, J. M. Bright1, S. M. Colley1, J. B. Hodgin3, O. Smithies3, and J. H. Tobias1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 ERbeta -/- mice previously generated by targeted gene deletion. Vehicle or 17beta -estradiol (E2) (range 4-4,000 µg · kg-1 · day-1) was administered to intact female ERbeta -/- 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 ERbeta -/- 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 ERbeta -/- 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 ERbeta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 ERbeta 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 ERbeta gene (ERbeta -/- mice) has provided an opportunity to explore the role of ERbeta in regulating skeletal responses to estrogen. On the basis of observations that cortical bone mass is increased in female animals, ERbeta has been suggested to act as a negative modulator of cortical bone growth (31). Although trabecular bone density in young adult female ERbeta -/- mice is unaffected (31), cancellous bone volume appears to be increased in older animals, associated with enhanced osteoblast activity (29), suggesting that ERbeta also suppresses osteoblast activity in cancellous bone. In the present investigation, we aimed to explore the role of ERbeta 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 ERbeta -/- animals.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental design. ERbeta -/- 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 ERbeta -/- 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 17beta -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.

Throughout, animals received a standard diet (Rat and Mouse Standard Diet; B&K, Humberside, UK) and water ad libitum and were kept on a 12:12-h light-dark cycle. The experimental duration was 28 days, tetracycline hydrochloride (25 mg/kg; Sigma) and calcein (30 mg/kg; Sigma) being injected intraperitoneally at 4 days and 1 day, respectively, before the mice were killed. At termination of the study, animals were killed by cervical dislocation, and long bones were removed for histomorphometric analysis. In addition, bone mineral density (BMD) was measured on whole tibiae by dual-X-ray absorptiometry with a PIXImus scanner (Lunar, Madison, WI) with small-animal software. All experimental procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

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 ERbeta -/- 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 ERbeta -/- 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 ERbeta -/- 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 ERbeta -/- 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 ERbeta -/- and control animals.


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Fig. 1.   Effects of 17beta -estradiol (E2) on histological appearance of proximal tibiae of wild-type and estrogen receptor (ER) beta -/- mice. Photomicrographs show longitudinal toluidine blue-stained sections of wild-type mice treated with vehicle (a), 40 µg/kg E2 (c), or 4,000 µg/kg E2 (e) and ERbeta -/- animals given vehicle (b), 40 µg/kg E2 (d), or 4,000 µg/kg E2 (f) (magnification ×25).



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Fig. 2.   Effects of varying doses of E2 at the proximal portion of the proximal tibial metaphysis (i.e., area 1) in wild-type () and ERbeta -/- () 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).



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Fig. 3.   Effects of varying doses of E2 at the distal portion of the proximal tibial metaphysis (i.e., area 2) in wild-type () and ERbeta -/- () 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: dlS/TV. B: BV/TV. Two-way ANOVA revealed a significant effect of dose (P < 0.0001).


                              
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Table 1.   Cancellous histomorphometry

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 ERbeta -/- 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 ERbeta -/- 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 ERbeta -/- mice, as illustrated by the significant interaction between estrogen dose and genotype.


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Fig. 4.   Effects of varying doses of E2 at the endocortical (Endo) surface in wild-type () and ERbeta -/- () 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: mineral apposition rate (MAR). B: double-labeled perimeter/bone perimeter (dlS/BS). Results show means ± SE. Two-way ANOVA revealed a significant effect of dose for MAR (P = 0.001) and of genotype for dlS/BS (P = 0.04).



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Fig. 5.   Effect of varying doses of E2 at the periosteal surface of wild-type () and ERbeta -/- () mice. Animals (4-7/group) were administered E2 0, 4, 40, 400, or 4,000 µg/kg by daily sc injection for 28 days. Results show means ± SE dlS/BS. Two-way ANOVA revealed a significant effect of dose (P = 0.003) and a significant interaction between dose and genotype (P = 0.05).

Cortical area and cross-sectional area were both significantly increased in ERbeta -/- mice compared with littermate controls (Table 2). Tibial BMD was also significantly higher in ERbeta -/- mice, but, unlike cortical area and cross-sectional area, this parameter additionally showed an increase after E2 treatment (Fig. 6).

                              
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Table 2.   Effects of varying doses of E2 on cortical areas in WT and ERbeta -/- mice



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Fig. 6.   Effect of varying doses of E2 on tibial bone mineral density (BMD) of wild-type () and ERbeta -/- () mice. Animals (4-7/group) were administered E2 0, 4, 40, 400, or 4,000 µg/kg by daily sc injection for 28 days. Results show means ± SE. Two-way ANOVA revealed a significant effect of dose (P < 0.0001) and genotype (P = 0.003).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We compared the osteogenic response of wild-type and ERbeta -/- 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 ERbeta -/- 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 ERbeta -/- 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 ERbeta -/- mice (29). Taken together, these findings suggest that ERbeta 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 ERalpha . Our preliminary findings from studies of ERalpha -/- mice are consistent with this suggestion.

Our observations raise the possibility that, in addition, ERbeta 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 ERbeta -/- 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 ERbeta -/- mice are partially protected against age-related bone loss suggest that, if anything, ERbeta acts as a negative regulator with respect to effects of physiological estrogen levels on bone formation, which may reflect inhibition by ERbeta of ERalpha expression or ERalpha -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 ERbeta -/- mice.

Cross-sectional area and cortical area of the tibial diaphysis were significantly higher in ERbeta -/- 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 ERbeta -/- 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 ERbeta 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 ERbeta -/- female mice is consistent with this possibility.

Taken together, our observations suggest that, whereas ERbeta 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 ERbeta 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 ERbeta 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 ERalpha or ERbeta .

In view of our findings that suggest that ERbeta 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 ERbeta -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 ERbeta -/- 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 ERbeta -/- mice have distinct skeletal phenotypes, were thought to reflect initial reports that ERbeta -/- mice as used in the present study express a mutant form of ERbeta that can bind estradiol but is unable to activate gene transcription (10). However, after reanalysis of variant ERbeta transcript sequences in ERbeta -/- 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 ERbeta protein being expressed. Nevertheless, although the reasons for the apparent difference in skeletal phenotype of these two lines of ERbeta -/- mice are unclear, it may be informative to repeat our investigations with the use of ERbeta -/- 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 ERbeta . Therefore, although ERbeta 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 ERbeta .


    ACKNOWLEDGEMENTS

This work was supported by the Nuffield Foundation (Oliver Bird Fund RHE/00031/G) and National Institutes of Health (GM-20069).


    FOOTNOTES

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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METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 283(4):E817-E823
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