Distinct effects of loss of classical estrogen receptor signaling versus complete deletion of estrogen receptor alpha on bone

Estrogen receptor (ER) α is a major regulator of bone metabolism which can modulate gene expression via a "classical" pathway involving direct DNA binding to estrogen-response elements (EREs) or via "non-classical" pathways involving protein-protein interactions. While the skeletal consequences of loss of ERE binding by ERα have been described, a significant unresolved question is how loss of ERE binding differs from complete loss of ERα. Thus, we compared the skeletal phenotype of wild-type (ERα(+/+)) and ERα knock out (ERα(-/-)) mice with that of mice in which the only ERα present had a knock-in mutation abolishing ERE binding (non-classical ERα knock-in [NERKI], ERα(-/NERKI)). All three groups were in the same genetic background (C57BL/6). As compared to both ERα(+/+) and ERα(-/-) mice, ERα(-/NERKI) mice had significantly reduced cortical volumetric bone mineral density and thickness at the tibial diaphysis; this was accompanied by significant decreases in periosteal and endocortical mineral apposition rates. Colony forming unit (CFU)-fibroblast, CFU-alkaline phosphatase, and CFU-osteoblast numbers were all increased in ERα(-/-) compared to ERα(+/+) mice, but reduced in ERα(-/NERKI) mice compared to the two other groups. Thus, using mice in identical genetic backgrounds, our data indicate that the presence of an ERα that cannot bind DNA but can function through protein-protein interactions may have more deleterious skeletal effects than complete loss of ERα. These findings suggest that shifting the balance of classical versus non-classical ERα signaling triggers pathways that impair bone formation. Further studies defining these pathways may lead to novel approaches to selectively modulate ER signaling for beneficial skeletal effects.     

Mechanical strain activates estrogen response elements in bone cells

The involvement of the estrogen receptor in the early responses of bone cells to mechanical strain was investigated by subjecting subconfluent monolayer cultures of ROS.SMER #14 cells (ROS 17/2.8 cells stably transfected with additional ER alpha) to 17 beta-estradiol or a single short period of dynamic mechanical strain (600 cycles, 1 Hz). The basal proliferation rate of ROS.SMER #14 cells was similar to ROS 17/2.8 cells, whose proliferative responsiveness to strain and estrogen is similar to that of primary cultures of rat long bone-derived osteoblasts. At peak strains of 3400 mu epsilon, strain-related proliferation in ROS.SMER #14 cells was 1.4 times that of ROS 17/2.8 cells. At 10(-8) mol/L, 17 beta-estradiol-related proliferation was nearly twice greater. The ROS.SMER #14 cells were transiently transfected with an estrogen-responsive reporter, 2ERE-pS2-CAT, containing two consensus estrogen response elements (ERE) linked to a chloroamphenicol acetyl transferase gene. Strain increased normalized ERE-CAT activity threefold and estradiol (10(-8) mol/L) sixfold. Both strain-related and estradiol-related increases in proliferation and ERE-CAT activity were blocked by the estrogen antagonist ICI 182,780 (10(-6) mol/L). These data show that strain as well as estrogen stimulates increased proliferation in ROS 17/2.8 cells and increased ER alpha-related ERE activity in ROS cells transfected with ER alpha. Proliferation is greater in the cells with more estrogen receptors. Both strain- and estrogen-related proliferation and ERE activity are blocked by the estrogen antagonist ICI 182,780. This indicates that ROS cells' early responses to mechanical strain involve ER alpha and estrogen-responsive genes.     

Estrogen receptor‐α is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving its activation function 1 but not 2

Estrogen receptor‐α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene‐targeted mouse models with (1) a complete ERα inactivation (ERα^?/?), (2) specific inactivation of activation function 1 (AF‐1) in ERα (ERαAF‐1⁰), or (3) specific inactivation of ERαAF‐2 (ERαAF‐2⁰) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα^?/? mice displayed a severely reduced osteogenic response to loading with changes in cortical area (?78% ± 15%, p < 0.01) and periosteal BFR (?81% ± 9%, p < 0.01) being significantly lower than in wild‐type (WT) mice. ERαAF‐1⁰ mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area ?40% ± 11%, p < 0.05 and periosteal BFR ?41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF‐2⁰ mice. Mechanical loading of transgenic estrogen response element (ERE)‐luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE‐mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving AF‐1 but not AF‐2. © 2013 American Society for Bone and Mineral Research     

Regulation of BMP-induced ectopic bone formation by Ahsg.

alpha2-HS-glycoprotein (Ahsg), also known as fetuin is a serum and bone resident glycoprotein, which binds to TGF-beta superfamily members including bone morphogenetic proteins (BMP) and inhibits dexamethasone-induced osteogenesis in bone marrow cultures in vitro. Here we demonstrate that Ahsg reduces cytokine binding to its cognate receptor in HOS osteocyte cells and suppresses intracellular signaling, while in vivo, we test the hypothesis that Ahsg-deficient mice are hyper-responsive to BMP-induced osteogenesis. Human native BMP was implanted into the hindquarter muscles of Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) mice and 4 weeks later, ossicle formation was analyzed by radiography, bone density scanning (DEXA) and histomorphometry. Alkaline phosphatase (AP) activity was measured in ossicles as a marker for bone cell differentiation, and was significantly higher in Ahsg(-/-) versus Ahsg(+/-) and/or Ahsg(+/+) mice. Ectopic ossicle size in the Ahsg(+/-) mouse was 4-fold greater than that in the wild type (Ahsg(+/+)), and intermediate to that shown in Ahsg(-/-) mouse. Bone mineral density (BMD) was lower in the Ahsg(-/+) and Ahsg(-/-) mice compared to Ahsg(+/+) littermates. The ratio of cortical to cancellous bone was found to be >2-fold higher in Ahsg(-/-) mouse in comparison to the Ahsg(+/+) mice with no significant change in the Ahsg(-/+) mouse. Finally, a significantly higher incidence of satellite ossification; small islands of immature bone, was shown in Ahsg(-/-) mice as compared to Ahsg(+/+) mice. Although Ahsg binds to TGF-beta/BMP and blocks receptor signalling, it may also sequester cytokines in matrix, thereby acting as a reservoir of osteoinductive activity when released. This may explain the non-linear relationship between ectopic bone formation characteristics and Ahsg(+/+), Ahsg(+/-) and Ahsg(-/-) genotypes, although the increase in satellite bone formation might also explain this phenomenon. Our results suggest that Ahsg may be useful for prevention of the heterotopic ossification and the regulation of osteoinductive effects of BMP used with grafts.     

Is interaction between age-dependent decline in mechanical stimulation and osteocyte–estrogen receptor levels the culprit for postmenopausal-impaired bone formation?

Declining estrogen levels during menopause are widely considered to be a major cause of age-dependent bone loss, which is primarily manifested by increased bone resorption by osteoclasts. We present accumulating evidence supporting another aspect of metabolic bone loss, suggesting that the combined interaction between age-dependent factors, namely, estrogen deficiency and reduced day-by-day activity/mechanical stimulation, directly leads to a reduction in anabolic processes. Such decreased bone formation results in diminished bone strength and failure to maintain the load-bearing competence of a healthy skeleton and to postmenopausal osteoporosis disorder. Estrogen receptors (ERs), as mediators of estrogenic actions, are essential components of bone osteocyte and osteoblast mechano-adaptive responses. ER expression appears to be upregulated by adequate circulating estrogen levels. ERα signaling pathways participate in the mechanotransduction response through obligatory “non-genomic” actions that occur independently of estrogen binding to ER and by a potentially “genomic”, estrogen-dependent mode. The experimental data indicate that cross talk between the ERα-“non-genomic” and Wnt/β-catenin signaling pathways constitutes the major regulatory mechanism. This interaction uses mechanically and ER-induced prostaglandin E2 as a mediator for the downregulation of osteocyte production of sclerostin. Sclerostin suppression, in turn, is a central prerequisite for load-induced formation and mineralization of the bone matrix. It is therefore plausible that future strategies for preventing and treating postmenopausal osteoporosis may use estrogenic compounds (such as selective estrogen receptor modulators or phytoestrogens) with physical activity, to complement antiresorptive therapy, aimed at stopping further bone loss and possibly even reversing it by stimulation of bone gain.     

Female Mice Lacking Estrogen Receptor‐Alpha in Osteoblasts Have Compromised Bone Mass and Strength

Reduced bioavailability of estrogen increases skeletal fracture risk in postmenopausal women, but the mechanisms by which estrogen regulates bone mass are incompletely understood. Because estrogen signaling in bone acts, in part, through estrogen receptor alpha (ERα), mice with global deletion of ERα (ERαKO) have been used to determine the role of estrogen signaling in bone biology. These animals, however, have confounding systemic effects arising from other organs, such as increased estrogen and decreased insulin‐like growth factor 1 (IGF‐1) serum levels, which may independently affect bone. Mice with tissue‐specific ERα deletion in chondrocytes, osteoblasts, osteocytes, or osteoclasts lack the systemic effects seen in the global knockout, but show that presence of the receptor is important for the function of each cell type. Although bone mass is reduced when ERα is deleted from osteoblasts, no study has determined if this approach reduces whole bone strength. To address this issue, we generated female osteoblast‐specific ERαKO mice (pOC‐ERαKO) by crossing mice expressing a floxed ERα gene (ERα^fl/fl) with mice transgenic for the osteocalcin‐Cre promoter (OC‐Cre). Having confirmed that serum levels of estrogen and IGF‐1 were unaltered, we focused on relating bone mechanics to skeletal phenotype using whole bone mechanical testing, microcomputed tomography, histology, and dynamic histomorphometry. At 12 and 18 weeks of age, pOC‐ERαKO mice had decreased cancellous bone mass in the proximal tibia, vertebra, and distal femur, and decreased cortical bone mass in the tibial midshaft, distal femoral cortex, and L5 vertebral cortex. Osteoblast activity was reduced in cancellous bone of the proximal tibia, but osteoclast number was unaffected. Both femora and vertebrae had decreased whole bone strength in mechanical tests to failure, indicating that ERα in osteoblasts is required for appropriate bone mass and strength accrual in female mice. This pOC‐ERαKO mouse is an important animal model that could enhance our understanding of estrogen signaling in bone cells in vivo. © 2014 American Society for Bone and Mineral Research.     

Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency.

Bone turnover requires the interaction of several proteases during the resorption phase. Indirect evidence suggests that the plasminogen activator/plasmin pathway is involved in bone resorption and turnover, and recently we have shown that this cascade plays a role in the degradation of nonmineralized bone matrix in vitro. To elucidate the role of the plasminogen activator inhibitor 1 (PAI-1) in bone turnover in vivo, bone metabolism was analyzed in mice deficient in the expression of PAI-1 gene (PAI-1-/-) at baseline (8-week-old mice) and 4 weeks after ovariectomy (OVX) or sham operation (Sham) and compared with wild-type (WT) mice. PAI-1 inactivation was without any effect on bone metabolism at baseline or in Sham mice. However, significant differences were observed in the response of WT and PAI-1-/- mice to ovariectomy. The OVX WT mice showed, as expected, decreased trabecular bone volume (BV/TV) and increased osteoid surface (OS/BS) and bone formation rate (BFR), as assessed by histomorphometric analysis of the proximal tibial metaphysis. In contrast, no significant change in any of the histomorphometric variables studied was detected in PAI-1-/- mice after ovariectomy. As a result, the OVX PAI-1-/- had a significantly higher BV/TV, lower OS/BS, lower mineral apposition rate (MAR) and BFR when compared with the OVX WT mice. However, a comparable decrease in the cortical thickness was observed in OVX PAI-1-/- and WT mice. In addition, the cortical mineral content and density assessed in the distal femoral metaphysis by peripheral quantitative computed tomography (pQCT), decreased significantly after ovariectomy, without difference between PAI-1-/- mice and WT mice. In conclusion, basal bone turnover and bone mass are only minimally affected by PAI-1 inactivation. In conditions of estrogen deficiency, PAI-1 inactivation protects against trabecular bone loss but does not affect cortical bone loss, suggesting a site-specific role for PAI-1 in bone turnover.     

Mice with increased angiogenesis and osteogenesis due to conditional activation of HIF pathway in osteoblasts are protected from ovariectomy induced bone loss

Postmenopausal osteoporosis is characterized by a reduction in the numbers of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion suggesting a role of vascular component in the pathogenesis of osteoporosis. Previous studies have shown that bone formation and angiogenesis are positively coupled through activation of the hypoxia inducible factor (HIF1α) signaling pathway. Therefore, we hypothesized that mice with increased angiogenesis and osteogenesis due to activation of the HIF signaling pathway in osteoblasts, via osteoblast specific disruption of HIF degrading protein von Hippel-Lindau (VHL) (ΔVhl), are protected from ovariectomy induced bone loss. ΔVhl mice and control littermates were ovariectomized or sham operated and four weeks later bone quality was evaluated along with blood vessel formation. Trabecular and cortical bone volume was strikingly increased in ΔVhl mice along with blood vessel formation as compared to control littermates. In control mice, ovariectomy significantly decreased bone mineral density, deteriorated bone microarchitecture, and decreased mechanical strength compared to the sham operated control mice. This was accompanied with a significant decrease in blood vessel volume and expressions of HIF1α, HIF2α, and VEGF proteins at the distal femur in ovariectomized control mice. In contrast, ovariectomy in ΔVhl mice had absolutely no effect on either the blood vessel formation or the bone structural and mechanical quality parameters. These data indicate that activation of HIF signaling pathway in osteoblasts may prevent estrogen deficiency-induced bone loss and decrease in blood vessels in bone marrow.     

Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen.

The role of mechanical strain and estrogen status in regulating ERalpha levels in bone cells was studied in female rats. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulna's medial surface, where artificial loading stimulates reversal of resorption to formation. INTRODUCTION: Osteoporosis is the most widespread failure of bones' ability to match their architectural strength to their habitual load bearing. In men and women, the severity of bone loss is associated with bioavailability of estrogen. This association could result from the estrogen receptor (ER) involvement in bone cells' adaptive response to loading. MATERIALS AND METHODS: In vivo semiquantitative analysis of the amount of ERalpha protein per osteocyte was performed in immuno-cytochemically stained sections from control and loaded rat ulna, as well as tibias of ovariectomy (OVX) and sham-operated female rats. In vitro, the effect of exogenous estrogen (10(-8) M) and mechanical strain (3400 microepsilon, 1 Hz, 600 cycles) on the expression of ERalpha mRNA levels was assessed in ROS 17/2.8 cells in monolayers using real-time PCR and ER promoter activity. ERalpha translocation in response to exogenous estrogen and mechanical strain was assessed in both ROS 17/2.8 and MLO-Y4 cells. RESULTS: More than 90 percent of tibial osteocytes express ERalpha, the level/osteocyte being higher in cortical than cancellous bone. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas in the ulna habitual strain and that caused by artificial loading had only a small but positive effect, except on the medial surface, where loading stimulates reversal of resorption to formation. In unstimulated osteocytes and osteoblasts in situ, and osteocyte-like and osteoblast-like cells in vitro, ERalpha is predominantly cytoplasmic. In vitro, both strain and estrogen stimulate transient ERalpha translocation to the nucleus and transient changes in ERalpha mRNA. Strain but not estrogen also induces discrete membrane localization of ERalpha. CONCLUSIONS: Bone cells' responses to both strain and estrogen involve ERalpha, but only estrogen regulates its cellular concentration. This is consistent with the hypothesis that bone loss associated with estrogen deficiency is a consequence of reduction in ERalpha number/activity associated with lower estrogen concentration reducing the effectiveness of bone cells' anabolic response to strain.     

Continuous parathyroid hormone and estrogen administration increases vertebral cancellous bone volume and cortical width in the estrogen-deficient rat.

Generally, it is believed that intermittent administration of parathyroid hormone (PTH) has an anabolic effect on the skeleton, whereas continuous administration is catabolic. However, there is evidence that continuous exposure to PTH may have an anabolic effect, for example, in patients with mild primary hyperparathyroidism (PHPT). The possibility of delivering PTH continuously may have important implications for the treatment of osteoporosis. Furthermore, estrogen treatment may be useful in the medical management of PHPT. Therefore, we examined the skeletal effects of continuous administration of PTH, with or without estrogen, in the estrogen-deficient rat with established osteopenia. Forty 7-month-old SD rats were divided into four ovariectomy (OVX) groups and one sham-operated group. Eight weeks post-OVX, three groups received subcutaneous implants of Alzet mini pumps loaded with PTH(1-34) (30 microg/kg per day), 17beta-estradiol (10 microg/kg per day) pellet, or both PTH and 17beta-estradiol separately for 4 weeks. OVX and sham control groups were given the mini pumps loaded with vehicle. Two doses of calcein (10 mg/kg) were given subcutaneously to all rats 2 days and 8 days before death. Histomorphometry was performed on cancellous and cortical bone of the fourth lumbar vertebra. At 3 months, post-OVX rats displayed bone loss with high bone turnover. Estrogen reversed OVX-mediated high turnover without restoring cancellous bone volume (BV/TV). PTH infusion further increased bone turnover and partially restored BV/TV. However, PTH infusion increased cortical porosity. Estrogen inhibited PTH-mediated cancellous bone resorption and substantially increased BV/TV above sham control. The combined treatment was associated with a significant increase in peritrabecular fibrosis and woven bone formation. The combined treatment of PTH infusion and estrogen replacement enhanced cortical width but estrogen did not prevent the PTH-induced cortical tunneling. We conclude that continuous administration of PTH and estrogen increases cortical porosity but has substantial beneficial effects on vertebral cancellous bone volume and cortical width in OVX rats.     

Cellular distribution of estrogen receptor beta in neonatal rat bone

Estrogens affect bone metabolism, and ovariectomy of rats results in marked bone loss caused by stimulation of osteoclastic bone resorption. Estrogen receptors (ER) have been demonstrated in osteoblasts and bone marrow stromal cells, but their presence in osteoclasts is controversial. Until recently, only one type of ER (now renamed ERalpha) had been identified. After the discovery of a novel ER subtype (ERbeta), it became necessary to re-investigate the ER expression in human and rodent bone. In the present study we examined the expression of ER mRNA in neonatal rat bone. Expression of ER alpha and beta mRNA (RT-PCR) was evident in femurs of 3-week-old male and female rats. In situ hybridization histochemistry of femural bones with digoxigenin labelled riboprobes, as well as radioactively labeled riboprobes, revealed that ERbeta mRNA was predominantly expressed in osteoblasts covering the metaphyseal bone trabecular surface. The presence of ERbeta mRNA in osteoblasts of rat bone suggests that ERbeta is involved in the mechanism of action of estrogens in bone.     

雌激素对实验性骨质疏松症骨折愈合的影响

目的 研究不同剂量的雌激素对卵巢除后发生骨质疏检习股骨骨折愈合的影响。方法 成年ｂａｌｂ／ｃ小鼠卵巢除后３个月开始制作右股骨中段闭合骨折模型,同一天开始皮下注射高低两种剂量雌激素,小鼠于折骨后５、１０、１５、２０、３０天分批处死,采用Ｘ射线、骨痂称重、骨痂中钙盐沉积率测定、组织学检查、血清生化检测等方法研究雌激素对骨折愈合的影响,并和卵巢除组、假手术对照组进行比较。结果 卵巢除组骨痂较小,     

Interleukin-11 receptor signaling is required for normal bone remodeling.

IL-6 and -11 regulate bone turnover and have been implicated in estrogen deficiency-related bone loss. In this study, deletion of IL-11 signaling, but not that of IL-6, suppressed osteoclast differentiation, resulting in high trabecular bone volume and reduced bone formation. Furthermore, IL-11 signaling was not required for the effects of estradiol or estrogen deficiency on the mouse skeleton. INTRODUCTION: Interleukin (IL)-6 and -11 stimulate osteoclastogenesis and bone formation in vitro and have been implicated in bone loss in estrogen deficiency. Because of their common use of the gp130 co-receptor signaling subunit, the roles of these two cytokines are linked, and each may compensate for the absence of the other to maintain trabecular bone volume and bone cell differentiation. MATERIALS AND METHODS: To determine the interactions in bone between IL-11 and IL-6 in vivo and whether IL-11 is required for normal bone turnover, we examined the bone phenotype of mature male and female IL-11 receptor knockout mice (IL-11Ralpha1-/-) and compared with the bone phenotype of IL-6-/- mice and mice lacking both IL-6 and IL-11Ralpha. To determine whether IL-11 is required for the effects of estrogen on trabecular bone, mature IL-11Ralpha1-/- mice were ovariectomized and treated with estradiol. RESULTS: In both male and female IL-11Ralpha1-/- mice, trabecular bone volume was significantly higher than that of wildtype controls. This was associated with low bone resorption and low bone formation, and the low osteoclast number generated by IL-11Ralpha1-/- precursors was reproduced in ex vivo cultures, whereas elevated osteoblast generation was not. Neither trabecular bone volume nor bone turnover was altered in IL-6-/- mice, and compound IL-6-/- :IL-11Ralpha1-/- mice showed an identical bone phenotype to IL-11Ralpha1-/- mice. The responses of IL-11Ralpha1-/- mice to ovariectomy and estradiol treatment were the same as those observed in wildtype mice. CONCLUSIONS: IL-11 signaling is clearly required for normal bone turnover and normal trabecular bone mass, yet not for the effects of estradiol or estrogen deficiency on the skeleton. In the absence of IL-11Ralpha, increased trabecular bone mass seems to result from a cell lineage-autonomous reduction in osteoclast differentiation, suggesting a direct effect of IL-11 on osteoclast precursors. The effects of IL-11Ralpha deletion on the skeleton are not mediated or compensated for by changes in IL-6 signaling.     

Osteoblast-like cells from estrogen receptor alpha knockout mice have deficient responses to mechanical strain.

INTRODUCTION: In vivo, bones' osteogenic response to mechanical loading involves proliferation of surface osteoblasts. This response is replicated in vitro and involves ERK-mediated activation of the estrogen receptor (ER) alpha and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERalpha. MATERIALS AND METHODS: We have now investigated the mechanisms of ER involvement in osteoblast-like cells' early responses to strain by comparing the responses of primary cultures of these cells derived from homozygous ERalpha knockout (ERKO) mice (ERalpha-/-) with those from their wildtype (ERalpha+/+) and heterozygous (ERalpha+/-) littermates and from ER/beta knockout (BERKO) mice (ERbeta+/+, ERbeta+/-, and ERbeta-/-). RESULTS: Whereas ERalpha+/+, ERalpha+/-, ERbeta+/+, and ERbeta-/- cells proliferate in response to a single 10-minute period of cyclic strain, ERalpha-/- cells do not. Transfection of fully functional, but not mutant, ERalpha rescues the proliferative response to strain in these cells. The strain-related response of ERalpha-/- cells is also deficient in that they show no increased activity of an AP-I driven reporter vector and no strain-related increases in NO production. Their strain-related increase in prostacyclin production is retained. They proliferate in response to fibroblast growth factor-2 but not insulin-like growth factor (IGF)-I or IGF-II, showing the importance of ERalpha in the IGF axis and the ability of ERalpha-/- cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERalpha. CONCLUSIONS: These data indicate ERalpha's obligatory involvement in a number of early responses to mechanical strain in osteoblast-like cells, including those that result in proliferation. They support the hypothesis that reduction in ERalpha expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.     

The effect of in vivo mechanical loading on estrogen receptor alpha expression in rat ulnar osteocytes.

The presence of estrogen receptor alpha (ER alpha) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 microm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10-minute periods of cyclic loading producing peak strains of either -3000 (n = 3) or -4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 +/- 1.2% SEM of all osteocytes in each bone section were ER alpha positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ER alpha (7.5 +/- 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 +/- 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ER alpha was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain-responsive cells. These data are consistent with the hypothesis that ER alpha is involved in bone cells' responses to mechanical strain. High strains appear to decrease ER alpha expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ER alpha levels and therefore impair the capacity for appropriate adaptive remodeling.