Tamoxifen Stimulates Cancellous Bone Formation in Long Bones of Female Mice
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内分泌学杂志 2005年第3期
Department of Anatomy (M.J.P., S.G., T.W.), and Department of Clinical Science at South Bristol (J.H.T.), University of Bristol, Bristol BS2 8HW, United Kingdom
Address all correspondence and requests for reprints to: Dr. J. Tobias, Rheumatology Unit, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom. E-mail: Jon.Tobias@bristol.ac.uk.
Abstract
Selective estrogen receptor modulators (SERMs) have been developed as a means of targeting estrogen’s protective effect on the skeleton in the treatment of postmenopausal osteoporosis. Although it is well established that SERMs such as tamoxifen inhibit bone resorption in a similar manner to estrogen, whether this agent shares estrogen’s stimulatory action on bone formation is currently unclear. To address this question, we compared the effect of treatment for 28 d with 17?-estradiol (E2; 0.1, 1.0 mg/kg·d) and tamoxifen (0.1, 1.0, or 10 mg/kg·d) on cancellous bone formation at the proximal tibial metaphysis of intact female mice. E2 stimulated the formation of new cancellous bone throughout the metaphysis. A similar response was observed after administration of tamoxifen, the magnitude of which was approximately 50% of that seen after E2. As expected, E2 was found to suppress longitudinal bone growth, but in contrast, this parameter was stimulated by tamoxifen. We conclude that tamoxifen acts as an agonist with respect to estrogen’s stimulatory action on bone formation but as an antagonist in terms of estrogen’s inhibition of longitudinal growth, suggesting that the protective effect of SERMs on the skeleton is partly mediated by stimulation of osteoblast activity.
Introduction
ALTHOUGH IT IS well established that estrogen exerts an important protective effect on the skeleton, the mechanisms responsible for this action are still being clarified. In addition to suppression of bone resorption and bone turnover, it has been suggested that prevention of bone loss by estrogen is mediated in part by stimulation of osteoblast function (1). Consistent with this possibility, recent histomorphometric analyses of iliac crest bone biopsies demonstrate that estradiol implants stimulate osteoblast activity in estrogen-deficient women (2, 3). These findings suggest that estrogen replacement may provide the basis for anabolic therapy in osteoporosis, particularly if administered at relatively high dose. In light of recent findings with teriparatide, such an approach may prove more effective compared with conventional antiresorptive regimes (4). However, this strategy is limited by recent evidence that estrogen replacement exerts adverse effects outside the skeleton, which may compromise the safety of long-term treatment regimes (5).
Alternatively, it may be possible to exploit estrogen’s stimulatory action on osteoblast function through the use of estrogen antagonists with tissue selective effects on bone, i.e. SERMS [selective estrogen receptor (ER) modulators]. This class of compound has been developed as a means of targeting estrogen’s protective effect on the skeleton while lacking unwanted effects at other sites. For example, tamoxifen and raloxifene both share estrogen’s ability to increase bone mass and reduce fracture risk in postmenopausal women but act as estrogen antagonists in reproductive tissues (6, 7). In terms of the mechanisms by which SERMs exert their protective effect on bone, clinical studies and investigations based on animal models suggest that these agents share estrogen’s ability to suppress bone resorption and turnover (8, 9, 10, 11, 12, 13, 14).
In addition, SERMs may share estrogen’s stimulatory action on osteoblast activity and hence provide an alternative basis for anabolic therapy in osteoporosis. Consistent with this suggestion, we recently found that estrogen-induced bone formation as assessed in intact mice requires the activation function-1 domain of ER (15), which is thought to represent an important pathway by which tamoxifen regulates gene activity (16). In addition, SERMs such as tamoxifen and raloxifene have been reported to share estrogen’s stimulatory action on osteoblast proliferation and differentiation in vitro (17, 18). Furthermore, we and others (19, 20, 21, 22) have found that SERMs stimulate a range of ostogenic growth factor genes in osteoblast-like cultures. However, evidence that tamoxifen or other SERMs stimulate bone formation in vivo, either in man or investigations based on animal models, is currently lacking. Therefore, in the present study, we investigated whether tamoxifen is able to reproduce estrogen’s stimulatory action on osteoblast function as assessed in vivo. We used intact mice for this purpose, in which model estrogen induces the rapid deposition of new cancellous bone within long bone metaphyses (23, 24, 25).
Materials and Methods
Ten-week-old female CBA-1 mice from the University of Bristol breeding colony were separated into three groups of seven to eight animals, which received vehicle (0.2 ml corn oil; Sigma, Poole, Dorset, UK), or 17?-estradiol (E2; Sigma) 0.1 or 1.0 mg/kg·d, by sc injection for 28 d. Additionally, three groups of 14–16 animals received tamoxifen 0.1, 1.0, or 10.0 mg/kg·d (Ultrafine, Salisbury, Wiltshire, UK). Standard tamoxifen formulations contain a small proportion of E-isomer that may act as an estrogen agonist (26). Therefore, to explore the effect of contamination with tamoxifen E-isomer, each tamoxifen group was further subdivided into two equal groups, to receive standard or E-isomer-free formulations (E-isomer content 0.3 and 0.006%, respectively). Calcein (30 mg/kg; Sigma) and tetracycline hydrochloride (25 mg/kg; Sigma) were injected ip at 1 and 4 d before the mice were killed, respectively, to provide a double fluorochrome label. After mice were killed, tibiae were removed and processed for histomorphometric analysis. Throughout the experiment, animals received a standard diet (rat and mouse standard diet; B&K Ltd., Humberside, UK) and water ad libitum, and they were kept with a 12-h light, 12-h dark cycle for 28 d. Animals were killed by cervical dislocation, their uteri weighed, and femurs and tibiae removed for processing. All experimental procedures were performed under a designated UK home office license and complied with the guiding principles in the National Institutes of Health Guide for the Care and Use of Animals.
Left tibiae were processed for histomorphometric analysis. Tibiae were removed of soft tissue, fixed in 70% ethanol for 48 h, then dehydrated through a graded series of alcohols: 80% ethanol, 90% ethanol, 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 London Resin white hard grade (London Resin Co., Reading, UK). Resin blocks were sanded manually on a belt sander along the longitudinal plane of the tibiae until the center of the tibiae was reached. Longitudinal sections of the proximal tibial metaphysis were cut on a Reicher-Jung 2050 microtome (Heidelberg, Germany) with a D-profile tungsten carbide knife. For each tibia, four 7-μm sections were stained for 3 min with 1% toluidine blue in 0.01 M citrate phosphate buffer and mounted in DPX (distrene, plasticizer, xylene) (Raymond A. Lamb Ltd., East Sussex, UK). Similarly, four 10-μm sections were cut but mounted unstained in fluoromount (BDH, Laboratory Supplies, Poole, UK) for assessment by fluorescent microscopy.
Histomorphometric analysis of the proximal tibial metaphysis was performed by transmitted and epifluorescent microscopy using a microscope (Leica, Milton Keynes, Buckinghamshire, UK; DMRB) linked to a computer-assisted image analyzer (Osteomeasure; Osteometrics, Atlanta GA) via a high-resolution video camera (Sony, Thatcham, Berkshire, UK; 3-charge-coupled device). All sections were examined blind. For each animal, two nonconsecutive sections were measured. A 0.6-mm wide x 1.2-mm long rectangular sampling site was used, the proximal border of which was located 0.25 mm below the lowest part of the growth plate to exclude primary spongiosa. The sampling site was centered within the shaft with respect to the edges of the cortical bone for each section. Cancellous bone parameters were assessed on two nonconsecutive sections stained with toluidine blue measured at x200 magnification and expressed as a percentage of total tissue volume. Toluidine blue-stained sections were also used to measure growth plate width and proliferative zone width, based on five measurements taken at equal intervals across the whole of the growth plate (proliferative chondrocytes were defined as having a height < 7 μm).
Fluorochome measurements were made on two nonconsecutive 10-μm sections per animal at x200 (unless otherwise stated). The length of trabecular bone surface covered by double label was expressed with reference to the total tissue volume [tissue volume referent, double-labeled surface (dLS)/TV] and as a percentage of the total length of cancellous bone surface (cancellous surface referent, dLS/BS). The 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 the section). The bone formation rate (BFR) was obtained from the product of MAR and either dLS/TV or dLS/BS, giving BFR/TV or BFR/BS, respectively. Longitudinal growth rate was determined by measuring the distance between the tetracycline and calcein bands lying distal to the epiphyseal growth plate and dividing by the time interval between the administration of the two labels.
Because two-way ANOVA showed no differences between tamoxifen formulations for effects on any of the parameters evaluated, these results were pooled for subsequent analysis. Groups for each outcome measure were then analyzed by one-way ANOVA followed by Tukey’s multiple comparison posttest where the ANOVA P value was 0.05.
Results
No significant effect was observed of any treatment regime on body weight gain (Table 1). Treatment with E2 1.0 mg/kg·d led to a significant increase in uterine weight in intact female mice. Although no significant effect of tamoxifen on uterine weight was observed, our results were consistent with a small decrease in this parameter. Treatment with E2 led to the accumulation of cancellous bone throughout the proximal tibial metaphysis in a dose-responsive manner, as assessed by histological appearance of toluidine blue-stained sections (Fig. 1), and histomorphometric quantification of cancellous bone volume (BV/TV) (Fig. 2A). This gain in cancellous bone reflected increased osteogenic activity because it was associated with a proportionate increase in the extent of cancellous mineralizing surfaces (tissue referent; dLS/BV) (Figs. 2B and 3).
TABLE 1. Effect of E2 or tamoxifen on body weight gain and uterine weight
FIG. 1. Toluidine blue-stained section of the proximal tibial metaphysis, after daily injections of vehicle (A), E2 0.1 (B) or 1.0 mg/kg·d (C), or tamoxifen 0.1 (D), 1.0 (E) or 10 mg/kg·d (F), for 28 d in intact CBA-1 female mice. A dose-responsive accumulation of cancellous bone was observed after treatment with tamoxifen as well as E2. Magnification, x30.
FIG. 2. Effects of E2 and tamoxifen on cancellous bone volume (A; BV/TV) and cancellous bone mineralizing parameter, tissue volume referent (B; dLS/TV). Results show mean ± SEM at the proximal tibial metaphysis after daily injections of vehicle, E2 0.1 or 1.0 mg/kg·d, or tamoxifen 0.1, 1.0, and 10 mg/kg·d, for 28 d in intact CBA-1 female mice. , Tamoxifen; , E2. a, P < 0.01 vs. vehicle; b, P < 0.01 vs. E2 0.1; c, P < 0.02 vs. tamoxifen 0.1, 1.0 and 10; d, P < 0.05 vs. tamoxifen 0.1 and 1.0; and e, P < 0.05 vs. tamoxifen 0.1
FIG. 3. Unstained section of the proximal tibial metaphysis viewed under UV light, after daily injections of vehicle (A), E2 0.1 (B) or 1.0 mg/kg·d (C), or tamoxifen 0.1 (D), 1.0 (E), or 10 mg/kg·d (F), for 28 d in intact CBA-1 female mice. A dose-responsive increase in mineralizing cancellous bone surfaces was observed after treatment with tamoxifen as well as E2. Magnification, x120.
Treatment with tamoxifen increased cancellous bone volume and the extent of mineralizing surfaces at the proximal tibial metaphysis (Figs. 1–3). A significant gain in BV/TV was observed after administration of tamoxifen 1.0 and 10, of which the latter dose resulted in a similar increase to that seen after E2 0.1 (Fig. 2A). All doses of tamoxifen significantly increased dLS/TV, but the magnitude of the response remained significantly below that after E2 1.0 (Fig. 2B). The response to tamoxifen appeared to be dose responsive across the range tested because the gain in BV/TV was greater in mice receiving tamoxifen 10 compared with tamoxifen 0.1 and 1, and the increase in dLS/TV was higher in those receiving tamoxifen 1 and 10 compared with tamoxifen 0.1.
Results for cancellous mineralizing surfaces (bone surface referent; dLS/BS) were also consistent with an osteogenic response to E2 and tamoxifen because this parameter showed small increases in the majority of treatment groups, which reached significance in the case of animals receiving tamoxifen 0.1 and 1.0 (Table 2). In contrast, MAR was found to be significantly lower in mice receiving E2 1.0 compared with animals treated with vehicle or tamoxifen. Finally, E2 1.0 was associated with a significant reduction in longitudinal growth, whereas tamoxifen led to a dose-responsive increase in this parameter (Fig. 4A). This stimulation of growth rate in response to tamoxifen was associated with increases in width of the growth plate and proliferating zone (Fig. 4, B and C).
TABLE 2. Effect of E2 or tamoxifen on cancellous histomorphometric parameters at the proximal tibial metaphysis
FIG. 4. Effect of E2 or tamoxifen on (A) longitudinal growth rate (LGR), (B) growth plate width (GPW), and (C) proliferating zone width (PZW), at the proximal tibial metaphysis. Results show mean ± SEM after daily injections of vehicle, E2 0.1 or 1.0 mg/kg·d, or tamoxifen 0.1, 1.0, and 10 mg/kg·d, for 28 d in intact CBA-1 female mice. , Tamoxifen; , E2. a, P < 0.05 vs. vehicle; b, P < 0.005 vs. all other groups; c, P < 0.001 vs. E2 0.1 and E2 1.0; and d, P < 0.0002 vs. tamoxifen 0.1.
Discussion
E2 caused marked accumulation of cancellous bone within the metaphysis of long bones of intact female mice. In line with our previous findings, this response appeared to reflect the de novo formation of new cancellous mineralizing surfaces throughout the proximal tibial metaphysis, as demonstrated by an equivalent increase in the absolute extent of cancellous mineralizing surfaces (25). As we have previously reported, this action of estrogen appears to have a relatively wide dose response range because a maximal response occurred after a very high dose of E2 1.0 mg/kg·d, accompanied by the formation of a dense trabecular network throughout the metaphysis (27). Interestingly, tamoxifen induced a similar osteogenic response to E2 because accumulation of cancellous bone was observed in a dose-responsive manner across the range of doses tested, in association with an increase in the extent of cancellous mineralizing surfaces.
Our findings are consistent with previous observations that tamoxifen stimulates osteoblast proliferation and differentiation under in vitro conditions (17, 18), and enhances the activity of reporter constructs for osteogenic genes transfected into osteoblasts, such as bone morphogenetic protein-4 and -6 and Cbfa1 (19, 21, 22). In addition, a potential anabolic activity of tamoxifen under in vivo conditions was noted in male castrated mice after observations that tamoxifen treatment led to an increase in bone density to beyond levels in intact animals (28). The implication from our findings that tamoxifen shares estrogen’s action in stimulating cancellous bone formation in mouse long bones is also consistent with previous in vivo observations that tamoxifen is able to reproduce estrogen’s antiresorptive action in cancellous bone as assessed in ovariectomized rats (11).
On the other hand, tamoxifen did not appear to replicate estrogen’s actions at the growth plate, in light of our observation that longitudinal growth rate was stimulated by tamoxifen, in association with an increase in width of the growth plate and proliferating zone. In contrast, estrogen suppressed longitudinal growth rate, and although a significant decrease in width of the growth plate and proliferating zones was not observed in the present study, this change has previously been reported in other animal studies (29). Therefore, taken together, these findings suggest that tamoxifen inhibits the tendency for endogenous estrogen to suppress bone growth, although further studies are required to confirm this possibility, for example by repeating these using ovariectomized animals treated with tamoxifen with or without estradiol. Interestingly, this conclusion contrasts with recent findings that suggest that raloxifene reproduces estrogen’s tendency to suppress longitudinal growth in the rabbit (29).
Based on current hypotheses of SERM action, any failure of tamoxifen to act as an estrogen agonist at the growth plate as opposed to other sites such as cancellous bone is likely to reflect significant differences in the expression, activity and subcellular localization of coactivators and corepressors involved in ER-dependent gene transcription in chondrocytes compared with other cell types in bone such as osteoblasts (30). Even in the context of cellular environments in which SERMs have the potential to act as estrogen agonists, such as cancellous bone, significant differences are likely to exist in the repertoire of genes induced by SERMs and estrogen. A restricted action of tamoxifen may underlie our finding that the gain in cancellous bone induced by tamoxifen 10 mg/kg·d was approximately 50% less than that observed after E2 1.0 mg/kg·d.
Although the smaller response to tamoxifen compared with E2 may reflect a lower response ceiling due to failure to activate the full range of estrogen target genes, results for cancellous bone volume suggest that the maximal response to tamoxifen may not have been reached by the 10 mg dose, implying that tamoxifen may also have a lower potency than E2. Previous studies in osteoblast cultures suggest that tamoxifen has a similar potency to E2 as assessed in vitro, based on observations that tamoxifen exerts ER-dependent effects on target genes across a similar dose range to E2 (31). Therefore, any reduced potency of tamoxifen compared with E2 under in vivo conditions is likely to reflect differences in pharmacokinetics. For example, the activity of tamoxifen requires biotransformation to 4-hydroxy-tamoxifen, which process may vary between mouse strains. Therefore, to explore the relative contributions of differences in response ceiling and potency to the submaximal response to tamoxifen, further studies are required using a wider dose range of tamoxifen combined with measurement of serum levels of tamoxifen and 4-hydroxy-tamoxifen.
Although the increase in extent of double-labeled cancellous bone surfaces was significantly less after treatment with tamoxifen 0.1 compared with E2 0.1, at higher doses, dLS/TV was similar after treatment with both of these agents. Moreover, MAR was higher after treatment with tamoxifen compared with E2. This apparent tendency for cancellous bone volume to show a greater response to E2 relative to tamoxifen compared with fluorochrome-based indices may reflect the fact that E2 induces a more rapid as well as stronger osteogenic response compared with tamoxifen, suggesting that the rate of new cancellous bone formation in the groups receiving E2 may have started to trail off by the time this was assessed at the end of the experimental period.
Because estrogen and tamoxifen are both known to exert important inhibitory actions on bone resorption in growing animals, this antiresorptive action may increase bone mass of long bone metaphyses by prevention of resorption of the primary spongiosa. Therefore, the antiresorptive action of estrogen and tamoxifen, which was not evaluated in the present study, may have contributed to the gain in cancellous bone volume that we observed at the proximal tibial metaphysis. However, our previous time-course study demonstrated that de novo medullary bone formation is largely responsible for the gain in cancellous bone induced by estrogen in intact female mice under the experimental conditions employed in the present investigation (25).
Whether other SERMs share the ability of tamoxifen to stimulate cancellous bone formation under in vivo conditions is currently unclear. For example, previous studies indicate that raloxifene, droloxifene, and idoxifene share tamoxifen’s tendency to suppress bone resorption action as assessed in ovariectomized rats (10, 12, 14), but the effect of these agents in an in vivo anabolic model as used in the present study has not to our knowledge been examined previously. However, there is some evidence to suggest that there are certain subclasses of ER antagonist that possess significant antiresorptive activity as assessed in vivo but have relatively little tendency to stimulate bone formation. For example, clomiphene and ICI 182,780 have previously been reported to inhibit ovariectomy-induced bone loss in the rat despite being classified as relatively pure estrogen antagonists (13, 32), but in our hands they possess little or no anabolic activity in the mouse model (27, 33).
Although our results suggest that SERMs such as tamoxifen are able to stimulate cancellous bone formation in the long bones of intact female mice, whether our findings can be applied to humans is currently unclear. When given routinely in postmenopausal women, estrogen acts to suppress bone resorption rather than to stimulate bone formation (34). However, when administered by implant leading to high and sustained exposure, there is evidence of a modest increase in osteoblast activity, as reflected by measurement of wall thickness in iliac crest bone biopsies obtained from postmenopausal women (2, 35) and younger women with Turner’s syndrome (3). Equivalent doses of E2 to those used in the present study are likely to result in serum E2 levels well beyond those achieved after implant therapy, on the basis of our previous dose-response studies in which E2 levels were found to be at least 10-fold higher than in intact mice (27, 33).
On the other hand, the dose range of tamoxifen found to stimulate cancellous bone formation in ovary intact mice encompassed the usual therapeutic dose of this agent in man (0.3 mg/kg·d). Although this dose of tamoxifen has been found to increase bone mass in postmenopausal women (7), a decrease in bone mass was observed in premenopausal women (36), presumably reflecting antagonism of endogenous estrogens. The latter finding raises the possibility that tamoxifen has less tendency to stimulate bone formation in man compared with animal models such as mice, which may reflect differences in pharmacokinetics between these two species, or in the cellular mechanisms involved in stimulation of bone formation in response to estrogen or SERMs. For example, our previous studies suggest that estrogen-induced bone formation in mice is largely mediated by the generation of early osteoblast precursors within the marrow cavity (37); whether the increase in wall thickness after estradiol implants in postmenopausal women reflects an equivalent action on recruitment of osteoblast precursors, or whether alternative mechanisms such as inhibition of osteoblast apoptosis are responsible as previously suggested (38), is currently unclear. Nevertheless, to the extent that the experimental model on which the present project is based holds some relevance for man, our findings raise the intriguing possibility that SERMs provide a basis for anabolic therapy in the treatment of osteoporosis.
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Address all correspondence and requests for reprints to: Dr. J. Tobias, Rheumatology Unit, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom. E-mail: Jon.Tobias@bristol.ac.uk.
Abstract
Selective estrogen receptor modulators (SERMs) have been developed as a means of targeting estrogen’s protective effect on the skeleton in the treatment of postmenopausal osteoporosis. Although it is well established that SERMs such as tamoxifen inhibit bone resorption in a similar manner to estrogen, whether this agent shares estrogen’s stimulatory action on bone formation is currently unclear. To address this question, we compared the effect of treatment for 28 d with 17?-estradiol (E2; 0.1, 1.0 mg/kg·d) and tamoxifen (0.1, 1.0, or 10 mg/kg·d) on cancellous bone formation at the proximal tibial metaphysis of intact female mice. E2 stimulated the formation of new cancellous bone throughout the metaphysis. A similar response was observed after administration of tamoxifen, the magnitude of which was approximately 50% of that seen after E2. As expected, E2 was found to suppress longitudinal bone growth, but in contrast, this parameter was stimulated by tamoxifen. We conclude that tamoxifen acts as an agonist with respect to estrogen’s stimulatory action on bone formation but as an antagonist in terms of estrogen’s inhibition of longitudinal growth, suggesting that the protective effect of SERMs on the skeleton is partly mediated by stimulation of osteoblast activity.
Introduction
ALTHOUGH IT IS well established that estrogen exerts an important protective effect on the skeleton, the mechanisms responsible for this action are still being clarified. In addition to suppression of bone resorption and bone turnover, it has been suggested that prevention of bone loss by estrogen is mediated in part by stimulation of osteoblast function (1). Consistent with this possibility, recent histomorphometric analyses of iliac crest bone biopsies demonstrate that estradiol implants stimulate osteoblast activity in estrogen-deficient women (2, 3). These findings suggest that estrogen replacement may provide the basis for anabolic therapy in osteoporosis, particularly if administered at relatively high dose. In light of recent findings with teriparatide, such an approach may prove more effective compared with conventional antiresorptive regimes (4). However, this strategy is limited by recent evidence that estrogen replacement exerts adverse effects outside the skeleton, which may compromise the safety of long-term treatment regimes (5).
Alternatively, it may be possible to exploit estrogen’s stimulatory action on osteoblast function through the use of estrogen antagonists with tissue selective effects on bone, i.e. SERMS [selective estrogen receptor (ER) modulators]. This class of compound has been developed as a means of targeting estrogen’s protective effect on the skeleton while lacking unwanted effects at other sites. For example, tamoxifen and raloxifene both share estrogen’s ability to increase bone mass and reduce fracture risk in postmenopausal women but act as estrogen antagonists in reproductive tissues (6, 7). In terms of the mechanisms by which SERMs exert their protective effect on bone, clinical studies and investigations based on animal models suggest that these agents share estrogen’s ability to suppress bone resorption and turnover (8, 9, 10, 11, 12, 13, 14).
In addition, SERMs may share estrogen’s stimulatory action on osteoblast activity and hence provide an alternative basis for anabolic therapy in osteoporosis. Consistent with this suggestion, we recently found that estrogen-induced bone formation as assessed in intact mice requires the activation function-1 domain of ER (15), which is thought to represent an important pathway by which tamoxifen regulates gene activity (16). In addition, SERMs such as tamoxifen and raloxifene have been reported to share estrogen’s stimulatory action on osteoblast proliferation and differentiation in vitro (17, 18). Furthermore, we and others (19, 20, 21, 22) have found that SERMs stimulate a range of ostogenic growth factor genes in osteoblast-like cultures. However, evidence that tamoxifen or other SERMs stimulate bone formation in vivo, either in man or investigations based on animal models, is currently lacking. Therefore, in the present study, we investigated whether tamoxifen is able to reproduce estrogen’s stimulatory action on osteoblast function as assessed in vivo. We used intact mice for this purpose, in which model estrogen induces the rapid deposition of new cancellous bone within long bone metaphyses (23, 24, 25).
Materials and Methods
Ten-week-old female CBA-1 mice from the University of Bristol breeding colony were separated into three groups of seven to eight animals, which received vehicle (0.2 ml corn oil; Sigma, Poole, Dorset, UK), or 17?-estradiol (E2; Sigma) 0.1 or 1.0 mg/kg·d, by sc injection for 28 d. Additionally, three groups of 14–16 animals received tamoxifen 0.1, 1.0, or 10.0 mg/kg·d (Ultrafine, Salisbury, Wiltshire, UK). Standard tamoxifen formulations contain a small proportion of E-isomer that may act as an estrogen agonist (26). Therefore, to explore the effect of contamination with tamoxifen E-isomer, each tamoxifen group was further subdivided into two equal groups, to receive standard or E-isomer-free formulations (E-isomer content 0.3 and 0.006%, respectively). Calcein (30 mg/kg; Sigma) and tetracycline hydrochloride (25 mg/kg; Sigma) were injected ip at 1 and 4 d before the mice were killed, respectively, to provide a double fluorochrome label. After mice were killed, tibiae were removed and processed for histomorphometric analysis. Throughout the experiment, animals received a standard diet (rat and mouse standard diet; B&K Ltd., Humberside, UK) and water ad libitum, and they were kept with a 12-h light, 12-h dark cycle for 28 d. Animals were killed by cervical dislocation, their uteri weighed, and femurs and tibiae removed for processing. All experimental procedures were performed under a designated UK home office license and complied with the guiding principles in the National Institutes of Health Guide for the Care and Use of Animals.
Left tibiae were processed for histomorphometric analysis. Tibiae were removed of soft tissue, fixed in 70% ethanol for 48 h, then dehydrated through a graded series of alcohols: 80% ethanol, 90% ethanol, 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 London Resin white hard grade (London Resin Co., Reading, UK). Resin blocks were sanded manually on a belt sander along the longitudinal plane of the tibiae until the center of the tibiae was reached. Longitudinal sections of the proximal tibial metaphysis were cut on a Reicher-Jung 2050 microtome (Heidelberg, Germany) with a D-profile tungsten carbide knife. For each tibia, four 7-μm sections were stained for 3 min with 1% toluidine blue in 0.01 M citrate phosphate buffer and mounted in DPX (distrene, plasticizer, xylene) (Raymond A. Lamb Ltd., East Sussex, UK). Similarly, four 10-μm sections were cut but mounted unstained in fluoromount (BDH, Laboratory Supplies, Poole, UK) for assessment by fluorescent microscopy.
Histomorphometric analysis of the proximal tibial metaphysis was performed by transmitted and epifluorescent microscopy using a microscope (Leica, Milton Keynes, Buckinghamshire, UK; DMRB) linked to a computer-assisted image analyzer (Osteomeasure; Osteometrics, Atlanta GA) via a high-resolution video camera (Sony, Thatcham, Berkshire, UK; 3-charge-coupled device). All sections were examined blind. For each animal, two nonconsecutive sections were measured. A 0.6-mm wide x 1.2-mm long rectangular sampling site was used, the proximal border of which was located 0.25 mm below the lowest part of the growth plate to exclude primary spongiosa. The sampling site was centered within the shaft with respect to the edges of the cortical bone for each section. Cancellous bone parameters were assessed on two nonconsecutive sections stained with toluidine blue measured at x200 magnification and expressed as a percentage of total tissue volume. Toluidine blue-stained sections were also used to measure growth plate width and proliferative zone width, based on five measurements taken at equal intervals across the whole of the growth plate (proliferative chondrocytes were defined as having a height < 7 μm).
Fluorochome measurements were made on two nonconsecutive 10-μm sections per animal at x200 (unless otherwise stated). The length of trabecular bone surface covered by double label was expressed with reference to the total tissue volume [tissue volume referent, double-labeled surface (dLS)/TV] and as a percentage of the total length of cancellous bone surface (cancellous surface referent, dLS/BS). The 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 the section). The bone formation rate (BFR) was obtained from the product of MAR and either dLS/TV or dLS/BS, giving BFR/TV or BFR/BS, respectively. Longitudinal growth rate was determined by measuring the distance between the tetracycline and calcein bands lying distal to the epiphyseal growth plate and dividing by the time interval between the administration of the two labels.
Because two-way ANOVA showed no differences between tamoxifen formulations for effects on any of the parameters evaluated, these results were pooled for subsequent analysis. Groups for each outcome measure were then analyzed by one-way ANOVA followed by Tukey’s multiple comparison posttest where the ANOVA P value was 0.05.
Results
No significant effect was observed of any treatment regime on body weight gain (Table 1). Treatment with E2 1.0 mg/kg·d led to a significant increase in uterine weight in intact female mice. Although no significant effect of tamoxifen on uterine weight was observed, our results were consistent with a small decrease in this parameter. Treatment with E2 led to the accumulation of cancellous bone throughout the proximal tibial metaphysis in a dose-responsive manner, as assessed by histological appearance of toluidine blue-stained sections (Fig. 1), and histomorphometric quantification of cancellous bone volume (BV/TV) (Fig. 2A). This gain in cancellous bone reflected increased osteogenic activity because it was associated with a proportionate increase in the extent of cancellous mineralizing surfaces (tissue referent; dLS/BV) (Figs. 2B and 3).
TABLE 1. Effect of E2 or tamoxifen on body weight gain and uterine weight
FIG. 1. Toluidine blue-stained section of the proximal tibial metaphysis, after daily injections of vehicle (A), E2 0.1 (B) or 1.0 mg/kg·d (C), or tamoxifen 0.1 (D), 1.0 (E) or 10 mg/kg·d (F), for 28 d in intact CBA-1 female mice. A dose-responsive accumulation of cancellous bone was observed after treatment with tamoxifen as well as E2. Magnification, x30.
FIG. 2. Effects of E2 and tamoxifen on cancellous bone volume (A; BV/TV) and cancellous bone mineralizing parameter, tissue volume referent (B; dLS/TV). Results show mean ± SEM at the proximal tibial metaphysis after daily injections of vehicle, E2 0.1 or 1.0 mg/kg·d, or tamoxifen 0.1, 1.0, and 10 mg/kg·d, for 28 d in intact CBA-1 female mice. , Tamoxifen; , E2. a, P < 0.01 vs. vehicle; b, P < 0.01 vs. E2 0.1; c, P < 0.02 vs. tamoxifen 0.1, 1.0 and 10; d, P < 0.05 vs. tamoxifen 0.1 and 1.0; and e, P < 0.05 vs. tamoxifen 0.1
FIG. 3. Unstained section of the proximal tibial metaphysis viewed under UV light, after daily injections of vehicle (A), E2 0.1 (B) or 1.0 mg/kg·d (C), or tamoxifen 0.1 (D), 1.0 (E), or 10 mg/kg·d (F), for 28 d in intact CBA-1 female mice. A dose-responsive increase in mineralizing cancellous bone surfaces was observed after treatment with tamoxifen as well as E2. Magnification, x120.
Treatment with tamoxifen increased cancellous bone volume and the extent of mineralizing surfaces at the proximal tibial metaphysis (Figs. 1–3). A significant gain in BV/TV was observed after administration of tamoxifen 1.0 and 10, of which the latter dose resulted in a similar increase to that seen after E2 0.1 (Fig. 2A). All doses of tamoxifen significantly increased dLS/TV, but the magnitude of the response remained significantly below that after E2 1.0 (Fig. 2B). The response to tamoxifen appeared to be dose responsive across the range tested because the gain in BV/TV was greater in mice receiving tamoxifen 10 compared with tamoxifen 0.1 and 1, and the increase in dLS/TV was higher in those receiving tamoxifen 1 and 10 compared with tamoxifen 0.1.
Results for cancellous mineralizing surfaces (bone surface referent; dLS/BS) were also consistent with an osteogenic response to E2 and tamoxifen because this parameter showed small increases in the majority of treatment groups, which reached significance in the case of animals receiving tamoxifen 0.1 and 1.0 (Table 2). In contrast, MAR was found to be significantly lower in mice receiving E2 1.0 compared with animals treated with vehicle or tamoxifen. Finally, E2 1.0 was associated with a significant reduction in longitudinal growth, whereas tamoxifen led to a dose-responsive increase in this parameter (Fig. 4A). This stimulation of growth rate in response to tamoxifen was associated with increases in width of the growth plate and proliferating zone (Fig. 4, B and C).
TABLE 2. Effect of E2 or tamoxifen on cancellous histomorphometric parameters at the proximal tibial metaphysis
FIG. 4. Effect of E2 or tamoxifen on (A) longitudinal growth rate (LGR), (B) growth plate width (GPW), and (C) proliferating zone width (PZW), at the proximal tibial metaphysis. Results show mean ± SEM after daily injections of vehicle, E2 0.1 or 1.0 mg/kg·d, or tamoxifen 0.1, 1.0, and 10 mg/kg·d, for 28 d in intact CBA-1 female mice. , Tamoxifen; , E2. a, P < 0.05 vs. vehicle; b, P < 0.005 vs. all other groups; c, P < 0.001 vs. E2 0.1 and E2 1.0; and d, P < 0.0002 vs. tamoxifen 0.1.
Discussion
E2 caused marked accumulation of cancellous bone within the metaphysis of long bones of intact female mice. In line with our previous findings, this response appeared to reflect the de novo formation of new cancellous mineralizing surfaces throughout the proximal tibial metaphysis, as demonstrated by an equivalent increase in the absolute extent of cancellous mineralizing surfaces (25). As we have previously reported, this action of estrogen appears to have a relatively wide dose response range because a maximal response occurred after a very high dose of E2 1.0 mg/kg·d, accompanied by the formation of a dense trabecular network throughout the metaphysis (27). Interestingly, tamoxifen induced a similar osteogenic response to E2 because accumulation of cancellous bone was observed in a dose-responsive manner across the range of doses tested, in association with an increase in the extent of cancellous mineralizing surfaces.
Our findings are consistent with previous observations that tamoxifen stimulates osteoblast proliferation and differentiation under in vitro conditions (17, 18), and enhances the activity of reporter constructs for osteogenic genes transfected into osteoblasts, such as bone morphogenetic protein-4 and -6 and Cbfa1 (19, 21, 22). In addition, a potential anabolic activity of tamoxifen under in vivo conditions was noted in male castrated mice after observations that tamoxifen treatment led to an increase in bone density to beyond levels in intact animals (28). The implication from our findings that tamoxifen shares estrogen’s action in stimulating cancellous bone formation in mouse long bones is also consistent with previous in vivo observations that tamoxifen is able to reproduce estrogen’s antiresorptive action in cancellous bone as assessed in ovariectomized rats (11).
On the other hand, tamoxifen did not appear to replicate estrogen’s actions at the growth plate, in light of our observation that longitudinal growth rate was stimulated by tamoxifen, in association with an increase in width of the growth plate and proliferating zone. In contrast, estrogen suppressed longitudinal growth rate, and although a significant decrease in width of the growth plate and proliferating zones was not observed in the present study, this change has previously been reported in other animal studies (29). Therefore, taken together, these findings suggest that tamoxifen inhibits the tendency for endogenous estrogen to suppress bone growth, although further studies are required to confirm this possibility, for example by repeating these using ovariectomized animals treated with tamoxifen with or without estradiol. Interestingly, this conclusion contrasts with recent findings that suggest that raloxifene reproduces estrogen’s tendency to suppress longitudinal growth in the rabbit (29).
Based on current hypotheses of SERM action, any failure of tamoxifen to act as an estrogen agonist at the growth plate as opposed to other sites such as cancellous bone is likely to reflect significant differences in the expression, activity and subcellular localization of coactivators and corepressors involved in ER-dependent gene transcription in chondrocytes compared with other cell types in bone such as osteoblasts (30). Even in the context of cellular environments in which SERMs have the potential to act as estrogen agonists, such as cancellous bone, significant differences are likely to exist in the repertoire of genes induced by SERMs and estrogen. A restricted action of tamoxifen may underlie our finding that the gain in cancellous bone induced by tamoxifen 10 mg/kg·d was approximately 50% less than that observed after E2 1.0 mg/kg·d.
Although the smaller response to tamoxifen compared with E2 may reflect a lower response ceiling due to failure to activate the full range of estrogen target genes, results for cancellous bone volume suggest that the maximal response to tamoxifen may not have been reached by the 10 mg dose, implying that tamoxifen may also have a lower potency than E2. Previous studies in osteoblast cultures suggest that tamoxifen has a similar potency to E2 as assessed in vitro, based on observations that tamoxifen exerts ER-dependent effects on target genes across a similar dose range to E2 (31). Therefore, any reduced potency of tamoxifen compared with E2 under in vivo conditions is likely to reflect differences in pharmacokinetics. For example, the activity of tamoxifen requires biotransformation to 4-hydroxy-tamoxifen, which process may vary between mouse strains. Therefore, to explore the relative contributions of differences in response ceiling and potency to the submaximal response to tamoxifen, further studies are required using a wider dose range of tamoxifen combined with measurement of serum levels of tamoxifen and 4-hydroxy-tamoxifen.
Although the increase in extent of double-labeled cancellous bone surfaces was significantly less after treatment with tamoxifen 0.1 compared with E2 0.1, at higher doses, dLS/TV was similar after treatment with both of these agents. Moreover, MAR was higher after treatment with tamoxifen compared with E2. This apparent tendency for cancellous bone volume to show a greater response to E2 relative to tamoxifen compared with fluorochrome-based indices may reflect the fact that E2 induces a more rapid as well as stronger osteogenic response compared with tamoxifen, suggesting that the rate of new cancellous bone formation in the groups receiving E2 may have started to trail off by the time this was assessed at the end of the experimental period.
Because estrogen and tamoxifen are both known to exert important inhibitory actions on bone resorption in growing animals, this antiresorptive action may increase bone mass of long bone metaphyses by prevention of resorption of the primary spongiosa. Therefore, the antiresorptive action of estrogen and tamoxifen, which was not evaluated in the present study, may have contributed to the gain in cancellous bone volume that we observed at the proximal tibial metaphysis. However, our previous time-course study demonstrated that de novo medullary bone formation is largely responsible for the gain in cancellous bone induced by estrogen in intact female mice under the experimental conditions employed in the present investigation (25).
Whether other SERMs share the ability of tamoxifen to stimulate cancellous bone formation under in vivo conditions is currently unclear. For example, previous studies indicate that raloxifene, droloxifene, and idoxifene share tamoxifen’s tendency to suppress bone resorption action as assessed in ovariectomized rats (10, 12, 14), but the effect of these agents in an in vivo anabolic model as used in the present study has not to our knowledge been examined previously. However, there is some evidence to suggest that there are certain subclasses of ER antagonist that possess significant antiresorptive activity as assessed in vivo but have relatively little tendency to stimulate bone formation. For example, clomiphene and ICI 182,780 have previously been reported to inhibit ovariectomy-induced bone loss in the rat despite being classified as relatively pure estrogen antagonists (13, 32), but in our hands they possess little or no anabolic activity in the mouse model (27, 33).
Although our results suggest that SERMs such as tamoxifen are able to stimulate cancellous bone formation in the long bones of intact female mice, whether our findings can be applied to humans is currently unclear. When given routinely in postmenopausal women, estrogen acts to suppress bone resorption rather than to stimulate bone formation (34). However, when administered by implant leading to high and sustained exposure, there is evidence of a modest increase in osteoblast activity, as reflected by measurement of wall thickness in iliac crest bone biopsies obtained from postmenopausal women (2, 35) and younger women with Turner’s syndrome (3). Equivalent doses of E2 to those used in the present study are likely to result in serum E2 levels well beyond those achieved after implant therapy, on the basis of our previous dose-response studies in which E2 levels were found to be at least 10-fold higher than in intact mice (27, 33).
On the other hand, the dose range of tamoxifen found to stimulate cancellous bone formation in ovary intact mice encompassed the usual therapeutic dose of this agent in man (0.3 mg/kg·d). Although this dose of tamoxifen has been found to increase bone mass in postmenopausal women (7), a decrease in bone mass was observed in premenopausal women (36), presumably reflecting antagonism of endogenous estrogens. The latter finding raises the possibility that tamoxifen has less tendency to stimulate bone formation in man compared with animal models such as mice, which may reflect differences in pharmacokinetics between these two species, or in the cellular mechanisms involved in stimulation of bone formation in response to estrogen or SERMs. For example, our previous studies suggest that estrogen-induced bone formation in mice is largely mediated by the generation of early osteoblast precursors within the marrow cavity (37); whether the increase in wall thickness after estradiol implants in postmenopausal women reflects an equivalent action on recruitment of osteoblast precursors, or whether alternative mechanisms such as inhibition of osteoblast apoptosis are responsible as previously suggested (38), is currently unclear. Nevertheless, to the extent that the experimental model on which the present project is based holds some relevance for man, our findings raise the intriguing possibility that SERMs provide a basis for anabolic therapy in the treatment of osteoporosis.
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