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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.

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.