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We recently found that estrogen receptor (ER) antagonists prevent high-dose estrogen from inducing the formation of new cancellous bone within the medullary cavity of mouse long bones. In the present investigation, we studied the role of specific ER subtypes in this response by examining whether this is impaired in female ERbeta(-/-) mice previously generated by targeted gene deletion. Vehicle or 17beta-estradiol (E(2)) (range 4-4,000 microg. 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. E(2) 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. E(2) 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 E(2) 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.

Parathyroid hormone (PTH) is thought to increase trabecular bone mass in postmenopausal women by stimulating osteoblast function. A similar action may contribute to estrogen's protective effect on the skeleton, which we have explored in female mice, in which estrogen induces an exaggerated osteogenic response. In the present investigation, we used this model to determine whether an interaction exists between stimulatory effects of PTH and estrogen on osteoblast function in cancellous bone. An initial dose response study was performed where PTH (hPTH, 1-38) was administered to ten-week-old intact female mice by daily sc injection for 28 days, at doses of 1, 10, 100 microg/kg. In a subsequent study, intact female mice were given PTH and/or 17beta-estradiol (E2) 10 and 40 microg/kg/day respectively. Femoral BMD was assessed by peripheral DXA (PIXImus), and histomorphometry was performed to analyse changes in cancellous and cortical bone. PTH caused a small gain in femoral BMD, and increased the extent of periosteal bone formation surfaces, but had relatively little effect on other skeletal parameters when given alone. As previously found, E2 produced a large increase in femoral BMD, stimulated cancellous and endocortical bone formation, but inhibited periosteal bone formation. In mice treated with combination therapy, a greater increase in femoral BMD was observed compared to that following treatment with either agent alone. No differences in indices of cancellous bone were found between animals treated with E2 compared to the combination group. However, cortical area and periosteal bone formation rate were significantly greater in the latter group. We conclude that PTH and E2 exert an additive effect on bone mass in long bones of female mice, possibly reflecting an ability of PTH to oppose E2-induced suppression of periosteal bone formation.

It is well recognized that high-dose estrogen induces a marked osteogenic response in long bones of female mice. In light of evidence which suggests that nitric oxide synthase (NOS) plays a role in regulation of osteoblast activity, we analyzed whether NOS is involved in mediating this response. Intact female mice were administered 17beta-estradiol (E(2)) either alone or in combination with N(G)-nitro-L-arginine methylester (L-NAME) or aminoguanidine (AG), over 24 days. The former inhibits both constitutive and inducible isoforms of NOS, whereas the latter is a selective inhibitor of inducible NOS. Bone mineral density (BMD) of the femur was subsequently measured by dual-energy X-ray absorptiometry (DXA), and histomorphometry performed at the proximal metaphysis on longitudinal tibial sections. As expected, E(2) given alone led to a marked accumulation of cancellous bone at the proximal tibial metaphysis, associated with a significant gain in femoral BMD, and an increase in cancellous mineralizing surfaces as assessed by histomorphometry. Neither L-NAME nor AG affected cancellous histomorphometric indices when given alone. However, when administered in combination with L-NAME, the magnitude of the skeletal response to E(2) was significantly reduced. The tendency for L-NAME to reduce estrogen-induced bone formation within the proximal tibial metaphysis was more marked distally compared with proximally. In contrast, AG showed no tendency to suppress the osteogenic response to E(2). Subsequently, we examined the effect of E(2) administration on expression within mouse femoral bone marrow of endothelial NOS (eNOS), which is the predominant constitutive isoform of NOS within bone. No change in eNOS mRNA levels was observed following E(2) administration, as assessed by reverse transcription-polymerase chain reaction (RT-PCR). Taken together, our results suggest that eNOS plays a role in mediating estrogen-induced bone formation in intact female mice, possibly as a consequence of posttranscriptional regulation of eNOS activity by estrogen.

Osteoblast differentiation under in vitro conditions is associated with increased expression of non-collagenous bone proteins including osteocalcin, osteopontin, and osteonectin, the exact function of which remain poorly understood. To determine whether these proteins play an important role in the formation of mineralised bone matrix by osteoblasts in vivo, we analysed the time-course of their expression during estrogen-induced osteogenesis in female mice, and compared this with the formation of new cancellous bone. Female mice were sacrificed prior to or following treatment with 17beta-estradiol for up to 32 days (500 microg/animal/week). Total RNA was extracted from femurs, and changes in expression of genes for a range of osteoblast-derived proteins assessed by Northern blot analysis. In parallel experiments, the time course of cancellous bone formation was determined by measuring bone mineral density (BMD) of the distal femur. Estrogen led to a rapid increase in BMD, which reached significance by Day 16. This was preceded by three-fold increases in expression of alkaline phosphatase (ALP) and type I collagen (COL I) at Days 8 and 12 respectively. In contrast, osteocalcin, osteopontin, and osteonectin expression showed no change during this initial period, although modest increases were observed at later times (i.e., Days 20 and 24). Our results suggest that osteocalcin, osteopontin, and osteonectin are not involved in the initial phase of the osteogenic response to estrogen, suggesting that these non-collagenous bone proteins do not play a direct role in the formation of mineralised bone matrix by osteoblasts in vivo., (Copyright 2001 Wiley-Liss, Inc.)

High-dose estrogen both stimulates new medullary bone formation and suppresses hematopoiesis in mouse long bones. To determine whether the latter response is a direct consequence of the former, we compared the time course of estrogen's effects on osteogenesis and hematopoietic bone marrow. Flow cytometry was employed to measure hematopoietic subpopulations in bone marrow from femurs of female mice killed at different times after commencing 0.5 mg estradiol/wk to each animal. Estrogen markedly reduced the number of leucocytes (CD11a positive), which had already diminished by 75% after 4 days and had virtually disappeared by 18 days. Specific populations showed a similar pattern of decline after estrogen, including B lymphocytes, monocytes, and endothelial cells. In contrast, the osteogenic precursor population showed a marked increase after estrogen treatment, as assessed by assaying alkaline phosphatase-positive colony-forming units (fibroblastic) ex vivo. However, this rise did not reach significance until 8 days after estrogen administration, suggesting that it follows rather than precedes estrogen's effects on hematopoiesis. We conclude that estrogen does not suppress hematopoiesis in mouse long bones as a direct consequence of its effects on osteogenesis.

Although estrogen is known to induce new bone formation in the long bones of female mice, this response is only thought to occur following administration of high doses, suggesting that it may not be mediated by a conventional estrogen receptor. To address this question further, we first examined the stereospecificity of this response by comparing the potency of 17beta-estradiol (E(2)) in stimulating cancellous bone formation at the proximal tibial metaphysis of intact female mice with that of the relatively inactive stereoisomer, 17alpha-estradiol (alphaE(2)). We found that E(2) was significantly more potent than alphaE(2), as assessed by histomorphometry. To provide further evidence for an estrogen-receptor-mediated process, we examined whether E(2)-induced osteogenesis in intact female mice could be inhibited by the estrogen receptor antagonist, ICI 182,780 (ICI). Although ICI itself had no effect on histomorphometric indices of the proximal tibial metaphysis when given alone, it significantly inhibited the osteogenic response to E(2). Finally, we examined the dose dependency of E(2)-induced osteogenesis at the proximal tibial metaphysis in intact mice. We found that E(2) stimulated cancellous bone formation in a dose-dependent manner over a wide dose range (i. e., 1-4000 microg/kg per day), with significant increases observed at doses of 4 microg/kg per day and beyond. Our results raise the possibility that estrogen-induced osteogenesis in the mouse represents an estrogen-receptor-mediated response that is not confined solely to supraphysiological estrogen levels.

We recently found that high-dose estrogen induces the formation of new sites of cancellous bone formation within the long bones of intact female mice. To examine whether prostaglandins play a role in mediating this response, we studied whether this is inhibited by coadministration of the cyclooxygenase inhibitor, indomethacin. Eight-week-old intact female mice were divided into four groups of ten, and administered vehicle, 17beta-estradiol (E2), at 500 microg/animal per week and/or indomethacin at 2 mg/kg per day. Animals were killed after treatment for 24 days, and histomorphometric indices subsequently analyzed on longitudinal sections of the proximal tibial metaphysis. As found previously, E2 treatment caused a striking increase in cancellous bone volume, associated with an equivalent increase in the extent of cancellous double-labeled surfaces. In mice treated with both indomethacin and E2, significant reductions in cancellous bone volume and cancellous double-labeled surfaces were observed as compared with animals treated with E2 alone. In contrast, indomethacin did not significantly influence these parameters when given alone. Subregional analysis within the proximal tibial metaphysis revealed that this inhibitory effect of indomethacin was more marked distally as compared with proximally, with the estrogen-induced gain in cancellous bone volume at these sites being reduced by 50% and 25%, respectively. We conclude that estrogen-induced osteogenesis in female mice is partially suppressed by treatment with indomethacin, suggesting that prostaglandin synthesis plays a significant role in mediating this response.

It is well recognized that, in the mouse, high-dose estrogen induces sclerosis within the shaft of long bones, an action that is largely thought to reflect increased osteoblastic cellular activity. We undertook to characterize this response in more detail, by performing a histologic analysis of the early changes induced by high-dose estrogen in the tibial cavity of young intact female mice. Female mice were sacrificed immediately before or 4, 8, 12, or 24 days after commencing subcutaneous injections of 17beta-estradiol (500 microg/animal/week), and longitudinal tibial sections were subsequently examined. Estrogen was found to cause a rapid gain in cancellous bone, with cancellous bone volume increasing by approximately 50% after 8 days, and by 5-fold after 24 days. Analysis of cancellous double-labeled surfaces revealed that this gain in bone reflected the emergence of new cancellous bone formation sites within the medullary cavity, rather than the reactivation and extension of formation over pre-existing bone surfaces. Comparison of the time course of these changes between proximal and distal regions of the proximal tibial metaphysis suggested that these new cancellous formation sites appear as a rapid wave extending distally from the secondary spongiosa. Alkaline phosphatase (ALP) immunocytochemistry revealed that, by 12 days after estrogen administration, a population of strongly ALP positive cells had appeared throughout the marrow cavity. We conclude that, at the proximal tibial metaphysis of female mice, estrogen-induced medullary sclerosis largely reflects a process of de novo medullary bone formation, possibly mediated by the generation of osteoblasts from bone marrow osteoprogenitor cells.

Although 17 beta-oestradiol (E2) is known to prevent bone loss, prolonged administration of E2 is unable to reverse this in female rats rendered osteopenic by ovariectomy. To determine whether this reflects a failure to replace other components of ovarian function involved in bone metabolism, we compared the effects of administering E2 to osteopenic ovariectomized (ovx) rats with those of ovarian transplantation. Ovariectomy was performed in female rats. After 13 weeks, by which time marked bone loss had occurred, one group of ovx animals received ovaries from donor rats, and, after a delay of 2 weeks to allow oestrus cycles to return, a further group received E2 5 micrograms.kg-1.day-1 for 9 weeks. The dose of E2 was chosen as that which in preliminary studies restored mean serum E2 levels to that of intact female rats. The study was terminated 24 weeks after ovariectomy. Both E2 and ovarian transplantation largely restored indices of oestrogenic exposure in ovx rats to those of sham-ovx animals. Animals receiving ovarian transplants also showed a small increase in serum progesterone and full restoration of serum testosterone. However, while ovarian transplantation also returned indices of cancellous bone metabolism to those of sham-ovx animals, there was little increase in bone volume. Interestingly, exogenous E2 caused a greater increase in cancellous bone volume than ovarian transplantation but also caused more marked suppression of bone formation, as assessed at the end of the study. In conclusion, exogenous E2 and ovarian transplantation exerted distinct effects on skeletal metabolism in osteopenic ovx rats, although the basis for this difference is currently unclear.

Although short-term administration of oestradiol-17 beta (OE2) stimulates cancellous bone formation in the rat, this is replaced by a tendency to suppression after prolonged treatment. Hence, in rats rendered osteopaenic by ovariectomy, OE2 administration fails either to induce a sustained increase in bone formation or to restore bone volume. A possible explanation for this failure is that OE2 also inhibits bone resorption, secondarily suppressing bone formation through coupling mechanisms. We therefore investigated whether the effects of OE2 treatment might be modified by intermittently stimulating bone resorption with retinoic acid (120mg/kg daily) for 4 out of every 20 days. We found, in a preliminary experiment using intact animals, that intermittent retinoic acid reduced cancellous bone volume, consistent with previously documented stimulation of bone resorption by retinoic acid. Rats were then rendered osteopaenic by ovariectomy, and given vehicle, retinoic acid and/or OE2. We found that animals treated with intermittent retinoic acid and OE2 showed a substantial increase in cancellous bone volume compared with ovariectomized animals treated with vehicle, retinoic acid alone or OE2 alone. Therefore, intermittent retinoic acid appears to cause a net increase in bone formation over resorption when given to ovariectomized animals in conjunction with OE2. We conclude that the effects of OE2 on cancellous bone are modified by intermittent treatment with retinoic acid, resulting in a substantial increase in bone volume.

Although estrogen is thought to protect the skeleton by inhibiting bone resorption, we have also found that in the rat, estrogen stimulates cancellous bone formation. However, the extent to which the various skeletal actions of estrogen are mediated by classical estrogen receptors remains unclear. Although estrogen receptor antagonists such as tamoxifen have been used to study this question, interpretation of the results is complicated by the fact that this agent also acts as a partial estrogen agonist. However, the recent development of estrogen antagonists devoid of agonist activity provides an opportunity to explore this question further. We, therefore, investigated the effect of administration of the pure estrogen antagonist ICI 182,780 on the skeleton of adult female rats. We found that ICI 182,780 reduced bone volume at the proximal tibial metaphysis by approximately 30%, associated with an increase in osteoclast surface. We then investigated the effect of ICI 182,780 on the anabolic action of estrogen. We used ovariectomized rats treated with 3-amino-1-hydroxypropylidene-1-bisphosphonate to inhibit bone resorption, thereby preventing any increase in bone formation as a result of the stimulation of bone resorption due to estrogen deficiency. 17 beta-Estradiol (1 micrograms/kg) stimulated cancellous bone formation in such animals by approximately 8-fold; this increase was abolished when ICI 182,780 was also given. In contrast, ICI 182,780 affected neither longitudinal nor periosteal tibial growth in either intact animals or ovariectomized rats given estradiol or vehicle. We conclude that ICI 182,780 reduces cancellous bone volume in the rat by antagonizing estrogen's actions on bone formation and resorption, suggesting that these processes are both mediated by classical estrogen receptors.

We have previously found that administration of oestradiol-17 beta (OE2) to intact adult female rats of 19 days stimulates cancellous bone formation. However, this effect is not observed following longer periods of OE2 treatment, suggesting that the responsiveness of the skeleton to oestrogen's anabolic action is reduced after prolonged administration. A possible explanation for this is that oestrogen also suppresses bone resorption, which is an important stimulus for bone formation. We therefore investigated the effect of omitting OE2 for short periods, on the proximal tibial metaphysis of intact female rats. We found that, unlike continuous treatment with OE2 (40 micrograms/kg) for 56 days, omission of OE2 for 4 days out of every 20 was associated with a significant increase in cancellous bone volume. Although continuous and intermittent OE2 were both associated with a reduction in osteoclast surface, a decrease in the proportion of double fluorochrome-labelled surface was only seen after continuous OE2 treatment. We then studied the effects of longer periods of OE2 omission by giving OE2 (40 micrograms/kg) for three repeated cycles of: (1) OE2 for 16 days/vehicle for 4 days, (2) OE2 for 12 days/vehicle for 8 days, (3) OE2 for 8 days/vehicle for 12 days, or (4) OE2 for 4 days/vehicle for 16 days. We found a significant increase in cancellous bone volume when OE2 was stopped for either 4 or 8 days at a time. However, longer periods of OE2 omission did not affect bone volume, possibly because these were associated with an increase in bone resorption and/or a reduction in bone formation during the OE2-free period.(ABSTRACT TRUNCATED AT 250 WORDS)

While the osteopenia associated with oestrogen deficiency is thought to arise from a relative defect in bone formation with respect to resorption, oestrogen administration itself leads to a decrease, rather than an increase, in bone formation. This decrease in bone formation, which arises from oestrogen's inhibitory effect on bone turnover, presumably masks any underlying tendency of oestrogen treatment towards stimulation of bone formation. To investigate this further, we have examined the early effect of discontinuing the administration of oestradiol-17 beta (OE2; 40 micrograms/kg) on bone formation indices in ovariectomized 13-week-old rats, before the turnover-induced increase in formation occurs. Histomorphometric indices were assessed at the proximal tibial metaphysis 0, 7, 10, 13 and 16 days following discontinuation of OE2 treatment. Measurements of body weight, uterine weight and longitudinal growth rate confirmed that there were rapid effects of OE2 deficiency on these parameters. We could detect no significant increase in bone resorption, as measured by osteoclast surface and number, until 16 days after ending treatment with OE2; this was coincidental with a reduction in bone volume. Shorter periods of OE2 deficiency were associated with a marked decrease in bone formation, as assessed by dynamic histomorphometric indices. This inhibition of bone formation was largely due to a reduction in double fluorochrome-labeled trabecular surfaces, which were decreased by approximately 70%. We conclude that ending OE2 administration in ovariectomized rats caused a striking decrease in trabecular bone formation, if such indices are assessed prior to the subsequent turnover-induced increase in formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen is generally considered to maintain bone mass through suppression of bone resorption. We have previously demonstrated that administration of pharmacologic doses of estrogen increases bone formation in ovary-intact rats. To assess the effects of physiological concentrations of estrogen on bone formation, estrogen was administered to ovariectomized rats in which bone resorption was suppressed by the bisphosphonate 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP). Animals receiving exogenous 17 beta-estradiol (E2) (1, 10, and 100 micrograms/kg daily for 17 d) showed a dose-dependent increase in trabecular bone volume of 1.9, 25.8, and 43.6%, respectively, compared with those rats treated with AHPrBP alone. The increase in bone volume was associated with an increase in bone formation in E2-treated animals, in which bone resorption had been almost completely suppressed by AHPrBP. Neither ovariectomy, AHPrBP, nor E2 treatment had a significant effect on the volume or rate of formation of cortical bone. Thus, the increased bone resorption, which is a consequence of estrogen-deficiency, entrains increased bone formation, which masks a simultaneous reduction in estrogen-dependent bone formation. Therefore, in addition to the nonspecific effect of estrogen to depress formation via coupling, we have identified a specific effect of estrogen to increase formation independent of coupling. Thus it appears that estrogen maintains bone volume not only through inhibition of bone resorption, but also through stimulation of bone formation.

Although sex hormones are important in the attainment and maintenance of bone mass, the mechanism by which they exert their effect is unknown. We therefore tested the effects of estradiol-17 beta, dihydrotestosterone, and progesterone on osteoclasts, the cells that resorb bone. Osteoclasts were disaggregated from neonatal rat long bones, and incubated with or without the addition of osteoblastic cells or osteoblast-like cell lines. Bone resorption was assessed by scanning electron microscopy as the extent of excavation of the bone surface after incubation. We found dihydrotestosterone (1-100 nmol/l) and progesterone (10-1000 nmol/l) to have no significant effect on bone resorption. By contrast, E2 (1 nmol/l) reduced bone resorption in osteoclast cultures to which osteoblasts had been added, by approximately 25%, although consistent inhibition with other concentrations (0.01, 0.1, 10 nmol/l) was not observed. To our surprise, E2 was also associated with a delayed, dose-responsive, stimulation of bone resorption, in the range 0.1-10 nmol/l, in osteoclast cultures free from added osteoblastic cells. Tamoxifen, which itself had no effect on bone resorption, appeared to antagonise these E2 effects. Although the physiological significance of the stimulatory effect is unclear, we hypothesize that its presence prevented us, and previous workers, from observing dose-responsive inhibition of bone resorption by E2 in vitro.

Although the effects of low concentrations of 17 beta-estradiol (E2) on bone formation and resorption are well described, little is known of the effects of E2 on bone at concentrations that circulate during pregnancy. We, therefore, investigated the effects of administration of high dose E2 to 3-month-old female Wistar rats on biochemical and histomorphometric indices of bone formation and resorption. Animals receiving exogenous E2 (4 mg/kg.day for 17 days; n = 9) showed a mean serum E2 concentration of 17.5 +/- 2.9 nM, compared with 0.6 +/- 0.2 nM in those receiving vehicle alone (n = 10). The bone formation rate, measured at the proximal tibial metaphysis after the administration of double fluorochrome labels, was greatly increased in the E2 group (13.6 +/- 2.0 x 10(-2) microns3/microns2.day) compared to controls (3.9 +/- 0.9), as was serum alkaline phosphatase (E2, 133.6 +/- 10.1 IU; controls, 87.5 +/- 5.5). This increase in the rate of bone formation was associated with a significant increase in trabecular bone volume. E2 treatment did not affect urinary hydroxyproline excretion or histomorphometric indices of bone resorption. These findings suggest that high concentrations of E2 strongly stimulate the formation of trabecular bone. This may represent an important mechanism by which calcium stores are accumulated during pregnancy in rats, in anticipation of the mineral requirements of lactation.