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Endocrinology Vol. 144, No. 5 1994-1999
Copyright © 2003 by The Endocrine Society

Estrogen Receptor-{alpha} Dependency of Estrogen’s Stimulatory Action on Cancellous Bone Formation in Male Mice

Kathleen E. McDougall, Mark J. Perry, Rachel L. Gibson, Shane M. Colley, Kenneth S. Korach and Jonathan H. Tobias

Academic Rheumatology (K.E.M., M.J.P., R.L.G., S.M.C., J.H.T.), University of Bristol, Bristol BS2 8HW, United Kingdom; and National Institute of Environmental Health Sciences (K.S.K.), National Institutes of Health, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Dr. J. H. Tobias, Rheumatology Unit, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom. E-mail: Jon.Tobias{at}bristol.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined whether estrogen receptor (ER){alpha} is required for estrogen to stimulate cancellous bone formation in long bones of male mice. 17ß-Estradiol (E2) was administered to ER{alpha}-/- male mice or wild-type (WT) littermate controls at 40, 400, or 4000 µg/kg by daily sc injection for 28 d and histomorphometric analysis performed at the distal femoral metaphysis. In WT mice, treatment with E2 (40 µg/kg·d) increased the proportion of cancellous bone surfaces undergoing mineralization and stimulated mineral apposition rate. In addition, higher doses of E2 induced the formation of new cancellous bone formation surfaces in WT mice. In contrast, E2 had little effect on any of these parameters in ER{alpha}-/- mice. Immunohistochemistry was subsequently performed using an ER{alpha}-specific C-terminal polyclonal antibody. In WT mice, ER{alpha} was expressed both by cancellous osteoblasts and a significant proportion of mononuclear bone marrow cells. Immunoreactivity was also observed in cancellous osteoblasts of ER{alpha}-/- mice, resulting from expression of the activation function-1-deficient 46-kDa ER{alpha} isoform previously reported to be expressed in normal osteoblasts and bones of ER{alpha}-/- mice. Taken together, our results suggest that estrogen stimulates bone formation in mouse long bones via a mechanism that requires the presence of full-length ER{alpha} possessing activation function-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN EXERTS A significant protective effect on the skeleton, the loss of which predisposes to the development of postmenopausal osteoporosis (1). This action has long been recognized to involve inhibition of osteoclastic bone resorption whereby estrogen acts to suppress bone turnover and prevent loss of cancellous bone (2, 3). In addition, recent observations suggest that estrogen, when administered at relatively high doses as estradiol implants, acts to stimulate osteoblast function at cancellous bone surfaces (4, 5). The latter effect may represent both direct actions of estrogen on osteoblasts involving the suppression of osteoblast apoptosis (6) and indirect effects mediated by locally produced growth factors in bone (7, 8).

The biological effects of estrogen are mediated by the estrogen receptor (ER), which exists in at least two distinct isoforms, ER{alpha} and ERß, both of which are expressed in bone cells at significant levels as assessed under in vitro and in vivo conditions (9, 10, 11, 12, 13, 14, 15, 16, 17). Recent in vitro studies suggest that ER{alpha} predominantly acts to mediate ligand-induced transcription, whereas ERß serves to modulate this response (18, 19). Consistent with this view, the finding of reduced bone mass in a man with ER{alpha} deficiency (20) suggests that ER{alpha} plays a central role in mediating the stimulatory action of estrogen on osteoblast function.

To date, analysis of the skeletal phenotype of ER{alpha}-/- mice has made a limited contribution to our understanding of the role of ER{alpha} in estrogen’s stimulatory action on osteoblast activity. For example, previous studies have found that rather than bone loss, ER{alpha}-/- mice demonstrate preserved or even increased cancellous bone mass (21, 22, 23). Although Sims et al. (21) found that indices of osteoblast function were reduced in male ER{alpha}-/- mice, this was associated with an increase in cancellous bone volume and a reduction in osteoclast surface and was thought to reflect reduced bone turnover rather than deficient ER{alpha}-dependent stimulation of osteoblast function.

We have used the mouse as an animal model to explore the stimulatory action of high-dose estrogen on cancellous bone formation, by analyzing changes in fluorochrome-based indices of bone formation in long bone sections following estrogen administration in intact female mice (24). In pharmacological studies, we confirmed that estrogen-induced cancellous bone formation in mouse long bones is ER dependent (25), but analysis of ERß-/- mice demonstrated that ERß is not required for this response (26). In the present study, we aimed to use the same approach to determine whether ER{alpha} is necessary for estrogen-induced bone formation, by analyzing this response in ER{alpha}-/- mice. Estradiol levels are grossly elevated in female ER{alpha}-/- mice (21), and, although this can be prevented by ovariectomy, the latter might engender further skeletal effects. Therefore, the present investigation used male ER{alpha}-/- mice, in view of previous findings that these show normal estradiol levels (21) and our unpublished observations that male mice show an equivalent cancellous bone response to estrogen to that in females as assessed by direct comparison of dose-response profiles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design
ER{alpha}-/- mice were generated at the National Institute of Environmental Health Sciences, back-crossed onto a C57Bl/6 genetic background, transferred to the University of Bristol animal facility, and crossed with wild-type C57Bl/6 mice from the local breeding stock (27). PCR-based genotyping was performed on DNA extracted from tail tips at 4–6 wk of age, based on previously published primer sets. Intact 14-wk-old male ER{alpha}-/- mice and age-matched littermate controls were subsequently administered vehicle [0.1 ml corn oil (Sigma, Poole, Dorset, UK)], or 17ß-estradiol (E2; Sigma) 40, 400, or 4000 µg/kg by daily sc injection (4–7 animals/group). This protocol was employed on the basis of our previous study in which we defined the dose responsiveness of estrogen-induced osteogenesis in intact female mice (25).

Throughout the study animals received a standard diet (Rat and Mouse Standard Diet, B&K Ltd., Humberside, UK) and water ad libitum and were kept with a cycle of 12-h light and 12-h darkness. The experimental duration was 28 d, with tetracycline hydrochloride (25 mg/kg; Sigma) and calcein (30 mg/kg; Sigma) being injected ip at 5 and 1 d, respectively, before the animals were killed. At termination of the study, animals were killed by cervical dislocation and long bones removed for histomorphometric analysis. All experimental procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and performed under appropriate licenses from the United Kingdom Home Office.

Histomorphometry
Femurs 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 three changes of 100% ethanol for 24 h each. Femurs were then cleared in chloroform for 24 h, placed for another 24 h in 100% ethanol and embedded without decalcification in LR White Hard Grade (London Resin Co., Reading, UK). Longitudinal sections of the distal portion of the femur were then prepared for histomorphometric analysis of the proximal tibial metaphysis, using a Reichert-Jung 2050 microtome (Cambridge Instruments GmbH, Heidelberg, Germany) with a "d" profile tungsten carbide knife; 7-µm sections were stained with 1% toluidine blue in 0.01 M citrate phosphate buffer for bone area measurement; 10-µm sections were mounted unstained in fluoromount (BDH Laboratory Supplies, Poole, UK) for assessment by fluorescent microscopy.

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. A standard area of 0.36 mm2 was used, the distal border of which was situated 0.25 mm above the growth plate to exclude the primary spongiosa. Cancellous bone area was expressed as a percentage of total tissue area (BV/TV).

The length of trabecular bone perimeter covered by double label 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 (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 (24). 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).

ER{alpha} immunostaining
Fourteen-week-old male wild-type (WT) and ER{alpha}-/- male mice were administered vehicle or E2, 4000 µg/kg, by weekly sc injection for 8 d. After the end of the experiment, tibiae were removed, freed from soft tissue, fixed in formol saline, and decalcified in EDTA. The metaphysis and diaphysis were separated, dehydrated, and paraffin embedded. Longitudinal sections (5 µm thick) were obtained at the proximal tibial metaphysis. Immunoreactivity for ER{alpha} was subsequently detected by incubating sections with a polyclonal rabbit antibody directed against a peptide mapping to the C terminus of murine ER{alpha} (MC-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Previous studies demonstrate that this antibody is not cross-reactive with ERß (28).

Sections were dewaxed, rehydrated, and rinsed in PBS with 0.2% Triton X-100, incubated with 10% normal goat serum (Sigma) in PBS with 0.2% Triton X-100 for 2 h to block any nonspecific binding of the secondary antibody, and then incubated with primary ER{alpha} antibody diluted 1:50 in normal goat serum overnight at 4 C. Sections were rinsed and incubated in alkaline phosphatase (ALP)-conjugated goat antirabbit IgG secondary antibody (Sigma) diluted 1:100 in PBS for 2 h at room temperature. The ALP conjugate was visualized using Fast Fast Red TR/Naphthol AS-MX tablet set (Sigma) containing 0.15 mg/ml levamisole to block endogenous ALP activity. Sections were rinsed, counterstained with hematoxylin, and mounted in Faramount aqueous mounting medium (DAKO Corp., Carpinteria, CA). To confirm specificity of the antibody, control sections were analyzed after preincubation with blocking peptide (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature.

Statistical analysis
Results are expressed as mean ± SEM. Two-way ANOVA was used to examine whether E2 treatment or genotype exerted statistically significant effects. The cut-off for statistical significance was taken as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histomorphometry
As previously found, treatment with high-dose E2 led to the appearance of new cancellous bone throughout the distal femoral metaphysis in WT male mice (Fig. 1Go). A similar response was not observed in ER{alpha}-/- animals. These findings were confirmed by histomorphometric analysis, which revealed an increase in cancellous bone volume following treatment with E2, 400 and 4000 µg/kg·d, within the distal femoral metaphysis of WT but not ER{alpha}-/- mice (Fig. 2AGo).



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  		Figure 1. Histological appearance of longitudinal sections of distal femoral metaphyses viewed by light microscopy. WT (A, C, E, and G) and ER{alpha}-/- (B, D, F, and H) mice were treated with vehicle (A and B) or E2 at 40 (C and D), 400 (E and F), or 4000 (G and H) µg/kg·d for 4 wk. Note the increase in cancellous bone formation following treatment with E2 in WT but not ER{alpha}-/- mice (x15).

 


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  		Figure 2. Cancellous bone histomorphometric indices as measured at the distal femoral metaphysis of WT (black squares) and ER-/- (white squares) mice, following treatment with vehicle or E2 at 40, 400, or 4000 µg/kg·d for 4 wk. Results show the following indices (mean ± SEM): cancellous bone volume (BV/TV; A); mineralizing surfaces (dlS/BS; B); mineralizing surfaces [tissue volume referent (dlS/BV; C]; and MAR (D). Two-way ANOVA revealed significant effects of genotype (P < 0.0001 for all indices), dose (P < 0.005 for BV/TV, dlS/BS, and dlS/BV), and genotype-dose interaction (P < 0.001 for BV/TV and dlS/BV, P < 0.05 for dlS/BS and MAR). *, P < 0.05 vs. vehicle (one-way ANOVA performed on data from WT animals).

 
Dynamic histomorphometry was employed to analyze the osteogenic response of cancellous bone to E2 in more detail. Following treatment with E2, an increase in the extent of cancellous mineralizing surfaces was observed in WT but not ER{alpha}-/- male mice (Fig. 3Go). Histomorphometric analysis demonstrated that in WT mice, treatment with E2, 40 µg/kg, acted to increase the proportion of cancellous bone surfaces undergoing mineralization (i.e. dlS/BS; Fig. 2BGo). In contrast, treatment with higher doses of E2 led to a significant increase in the absolute extent of mineralizing surfaces (i.e. dlS/TV; Fig. 2CGo), indicating the formation of new cancellous bone surfaces as previously observed following high-dose estrogen (24). In ER{alpha}-/- mice, relatively little change was observed in either dlS/BS or dlS/TV following administration of E2. An increase in MAR also contributed to the stimulatory action of E2 on bone formation in WT but not ER{alpha}-/- mice, following treatment with both low- and high-dose E2 (Fig. 2D).



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  		Figure 3. Mineralizing surfaces in longitudinal sections of distal femoral metaphyses viewed under UV light. WT (A, C, E, and G) and ER{alpha}-/- (B, D, F, and H) mice were treated with vehicle (A and B) or E2 at 40 (C and D), 400 (E and F), and 4000 µg/kg·d (G and H) for 4 wk. WT mice showed an increase in the extent of cancellous mineralizing surfaces following treatment with all doses of E2. An equivalent response was not observed in ER{alpha}-/- mice (x 50).

 
ER{alpha} immunostaining
We explored the role of ER{alpha} in regulating osteoblast function in cancellous bone by analyzing its expression in the proximal tibial metaphysis by immunohistochemistry. In WT vehicle-treated mice, ER{alpha} was found to be expressed by the majority of osteoblasts on cancellous bone surfaces (Fig. 4AGo). A significant number of bone marrow cells were also noted to express ER{alpha}, which tended to occur within groupings of positive cells randomly situated within the marrow cavity (Fig. 4BGo). In addition, megakaryocytes were consistently ER{alpha} positive. Little immunoreactivity was observed in the presence of blocking peptide (Fig. 4CGo).



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  		Figure 4. ER{alpha} expression in longitudinal sections of the proximal tibial metaphysis as assessed by light microscopy. WT and ER{alpha}-/- mice were treated with vehicle or E2, 4000 µg/kg·d, for 8 d. Sections from WT vehicle-treated mice showed ER{alpha} expression by the majority of osteoblasts on cancellous bone surfaces (A; arrows); ER{alpha} expression by megakaryocytes (B; black arrows) and mononuclear bone marrow cells (B; white arrows); and osteoblasts showed reduced immunostaining following preincubation with blocking peptide (C; arrows). In WT mice treated with E2, a substantial increase in number of cancellous osteoblasts was observed (D), the majority of which expressed ER{alpha} (arrows). In ER{alpha}-/- mice treated with vehicle (E) or E2 (F), comparable numbers of osteoblasts were observed with those in vehicle-treated WT animals, and the majority of osteoblasts expressed ER{alpha} (arrows; x250).

 
Following E2 treatment, a substantial increase in number of ER{alpha}-positive cancellous osteoblasts was seen in WT mice, whereas the degree and pattern of bone marrow expression was unchanged (Fig. 4DGo). Interestingly, the majority of cancellous osteoblasts in ER{alpha}-/- mice also demonstrated immunoreactivity to ER{alpha}, both in vehicle- and E2-treated animals (Fig. 4Go, E and F). Presumably the latter observations reflect the fact that an N-terminal-truncated ER{alpha} isoform also exists, which is known to be expressed in ER{alpha}-/- mice (29), and would have been detectable by the MC-20 antibody, which is directed against the ER{alpha} C terminus. Cells that expressed ER{alpha} generally demonstrated a combination of nuclear and cytoplasmic staining, with no differences in localization observed between WT and ER{alpha}-/- mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that different doses of E2 exerted distinct actions on osteoblast function in cancellous bone of male mice, all of which were abolished in ER{alpha}-/- animals. E2 as administered at the dose of 40 µg/kg·d increased MAR and the proportion of cancellous bone surfaces undergoing mineralization. These findings are consistent with the effects of E2 treatment on cancellous bone formation as previously documented in female rats (30) and reports that estradiol implants increase osteoblast lifespan and/or work rate in postmenopausal women as assessed by measurement of mean wall thickness (4, 5) and may reflect a tendency of E2 to suppress osteoblast apoptosis (6). Because the stimulatory effect of E2, 40 µg/kg·d, on osteoblast function was found to be absent in ER{alpha}-/- mice, we concluded that this action is ER{alpha} dependent. As well as stimulating mineral apposition rate, E2 at 400 and 4000 µg/kg·d induced the formation of new cancellous bone formation surfaces as reflected by an increase in the absolute extent of cancellous mineralization surfaces and a substantial gain in cancellous bone volume, as previously reported in female mice (24). This response to high-dose E2, which our previous observations suggest involves the generation of osteoblast precursors from osteoprogenitors within bone marrow (31, 32), was also abrogated in ER{alpha}-/- mice.

To our knowledge, the present findings are unique in that no previous study has directly examined whether estrogen’s stimulatory action on osteoblast function in cancellous bone is impaired following targeted gene deletion of ER{alpha}. Previous reports indicated that, whereas E2 as administered at levels similar to the 40-µg/kg dose in the present study increases cancellous bone volume in orchidectomized male mice, no response occurs in ER{alpha}-/- animals (22, 33). However, prevention of bone loss by estrogen following orchidectomy may partly reflect this hormone’s antiresorptive action, and because the latter studies did not analyze fluorochrome-based indices of cancellous bone formation, it is not possible to determine whether lack of estrogen’s stimulatory action on osteoblast function underlies these previous observations.

To explore the role of ER{alpha} in mediating the osteogenic response to estrogen, immunohistochemical studies were performed, which revealed that ER{alpha} is expressed at significant levels within cancellous bone by osteoblasts. These results are in keeping with previous findings that ER{alpha} is expressed by isolated osteoblasts as assessed in vitro (9, 10, 11, 12) and osteoblasts and bone marrow at cancellous-rich sites within neonatal human ribs (13). These findings may reflect the fact that direct ER{alpha}-dependent activation of osteoblasts contributes to estrogen-induced bone formation, which is consistent with a recent report that estrogen acts directly on osteoblasts to suppress their apoptosis (6). The observation that ER{alpha} showed cytoplasmic as well as nuclear staining in bone tissue sections is consistent with this mode of action, in view of the suggestion that this involves a nongenotropic pathway (6). Significant ER{alpha} expression was also found in megakaryocytes, as previously reported in human bone (34) and a subset of mononuclear bone marrow cells. The latter may have included stromal cells, which may also contribute to estrogen-induced bone formation in mouse long bones by stimulating the formation of osteoblast precursors in bone marrow following the release of osteogenic growth factors (8).

Interestingly, significant ER{alpha} immunoreactivity was observed in cancellous bone of ER{alpha}-/- mice with a similar intracellular and tissue distribution to that of vehicle-treated WT animals. This finding is consistent with recent reports that osteoblasts also express significant levels of an N-terminal-deficient 46-kDa isoform of ER{alpha} and that the latter is expressed in bones from ER{alpha}-/- mice as used in the present study (29). Presumably, ER{alpha} immunoreactivity that we observed in ER{alpha}-/- mice was related to the presence of this 46-kDa ER{alpha} isoform. The MC-20 antibody used in the present study was raised against a peptide mapping to the C-terminal of mouse ER{alpha} and is therefore expected to detect the 46-kDa ER{alpha} isoform in which the C-terminal is intact, although confirmatory studies are required to test this assumption. In view of the lack of estrogen response in ER{alpha}-/- mice, our results imply that estrogen-induced bone formation requires full-length ER{alpha}. This finding may represent an important role of the activation function (AF)-1 of ER{alpha}, which is deficient in the N-terminal-truncated 46-kDa isoform of ER{alpha} (29).

Our previous results, which suggest that ERß does not mediate the osteogenic response to estrogen, are consistent with an important role of AF-1 in bone formation (26) because ERß is also thought to be deficient in terms of its AF-1 function (35). This possibility is supported by findings with the estrogen antagonist, tamoxifen, which paradoxically acts as an estrogen agonist in bone (36), and stimulates bone formation in mouse long bones with a potency approaching that of E2 (our unpublished results). Because tamoxifen is thought to regulate gene activity through AF-1 (37), the latter domain is also likely to play a role in mediating ER{alpha}-dependent activation of bone formation in response to estrogen antagonists.

The absence of an osteogenic response of male ER{alpha}-/- mice to estrogen contrasts with a previous report that in female double-ER knockout mice generated by crossing ER{alpha}-/- animals as used in the present study with ERß-/- mice, ovariectomy leads to bone loss, which can be prevented by high-dose E2 (38). Prevention of ovariectomy-induced bone loss by estrogen is largely thought to reflect this hormone’s antiresorptive action (3). Therefore, taken with results of the present study, these findings suggest that whereas AF-1 is required for estrogen’s osteogenic action, estrogen’s antiresorptive effect is mediated by a distinct, AF-1-independent pathway.

Whether estrogen’s stimulatory action on osteoblast function in humans is also mediated by ER{alpha} is currently unclear. Estrogen’s tendency to stimulate cancellous bone formation in mouse long bones is somewhat exaggerated, compared with the response observed in postmenopausal women treated with estradiol implants (4, 5), and it is possible that certain species differences exist between the molecular pathways involved. Nevertheless, the observation that ER{alpha} deficiency in an adult male is associated with impaired acquisition of peak bone mass (20) supports the possibility that ER{alpha} is also required for estrogen’s stimulatory action on osteoblast function in humans. To the extent that our findings can be extrapolated to humans in this way, our results indicate that it may be possible to develop novel bone-forming therapies for postmenopausal osteoporosis, based on agents that target ER{alpha}/AF-1-dependent responses in osteoblasts.


   Footnotes
 
This work was supported by the Oliver Bird Fund, Nuffield Foundation.

Abbreviations: AF, Activation function; ALP, alkaline phosphatase; BV/TV, cancellous bone area expressed as a percentage of total tissue area; dlS/BS, percentage of the total length of cancellous bone perimeter; dlS/TV, tissue area referent; E2, 17ß-estradiol; ER, estrogen receptor; MAR, mineral apposition rate; WT, wild-type.

Received November 25, 2002.

Accepted for publication January 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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