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Postmenopausal osteoporosis represents a failure of the response by which bone cells adapt bone mass and architecture to be sufficiently strong to withstand loading without fracture. To address why this failure should be associated with oestrogen withdrawal, we investigated the ulna's adaptive response to mechanical loading in adult female mice lacking oestrogen receptor-alpha (ERalpha(-/-)), those lacking oestrogen receptor-beta (ERbeta(-/-)) and their wild-type littermates. In wild-type mice, short periods of physiologic cyclic compressive loading of the ulna in vivo over a 2-week period stimulates new bone formation. In ERalpha(-/-) and ERbeta(-/-) mice this osteogenic response was respectively threefold and twofold less (P<0.05). In vitro, primary cultures of osteoblast-like cells derived from these mice were subjected to a single short period of mechanical strain. Twenty-four hours after strain the number of wild-type cells was 61+/-25% higher than in unstrained controls (P<0.05), whereas in ERalpha(-/-) cells there was no strain-related increase in cell number. However, the strain-related response of ERalpha(-/-) cells could be partially rescued by transfection with functional human ERalpha (P<0.05). ERbeta(-/-) cells showed a 125+/-40% increase in cell number following strain. This was significantly greater than in wild types (P<0.05).These data support previous findings that functional ERalpha is required for the full osteogenic response to mechanical loading and particularly the stage of this response, which involves an increase in osteoblast number. ERbeta appears to depress the ERalpha-mediated strain-related increase in osteoblast number in vitro, but in female transgenic mice in vivo the constitutive absence of either ERalpha or ERbeta appears to diminish the osteogenic response to loading.

The presence of estrogen receptor α (ERα) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 μm 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 ERα 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 ERα (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 ERα 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 ERα is involved in bone cells' responses to mechanical strain. High strains appear to decrease ERα expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ERα levels and therefore impair the capacity for appropriate adaptive remodeling.

The presence of estrogen receptor α (ERα) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 μm 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 ERα 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 ERα (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 ERα 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 ERα is involved in bone cells' responses to mechanical strain. High strains appear to decrease ERα expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ERα levels and therefore impair the capacity for appropriate adaptive remodeling.

Bone cells' early responses to estrogen and mechanical strain were investigated in the ROS 17/2.8 cell line. Immunoblotting with antiphosphorylated estrogen receptor α (ER‐α) antibody showed that when these cells were exposed for 10 minutes to estrogen (10−8M) or a single period of cyclic dynamic strain (peak 3400 μϵ, 1Hz, 600 cycles), there was an increase in the intensity of a 66‐kDa band, indicating phosphorylation of ser122in the amino terminus of ER‐α. Increased phosphorylation was detected within 5 minutes of exposure to estrogen and 5 minutes after the end of the period of strain. Estrogen and strain also activated the mitogen‐activated protein kinase (MAPK) family member extracellular regulated kinase‐1 (ERK‐1). Increases in ERK activation coincided with increased ER‐α phosphorylation. Activation of ERK‐1 and the phosphorylation of ER‐α, by both estrogen and strain, were prevented by the MAP kinase kinase (MEK) inhibitor U0126 and the protein kinase A (PKA) inhibitor (PKI). These data support previous suggestions that resident bone cells' early responses to strain and estrogen share a common pathway, which involves ER‐α. This pathway also appears to involve PKA and ERK‐mediated phosphorylation of ser122within the amino terminus of ER‐α. Reduced availability of this pathway when estrogen levels are reduced could explain diminished effectiveness of mechanically related control of bone architecture after the menopause.

Bone cells' early responses to estrogen and mechanical strain were investigated in the ROS 17/2.8 cell line. Immunoblotting with antiphosphorylated estrogen receptor α (ER‐α) antibody showed that when these cells were exposed for 10 minutes to estrogen (10−8M) or a single period of cyclic dynamic strain (peak 3400 μϵ, 1Hz, 600 cycles), there was an increase in the intensity of a 66‐kDa band, indicating phosphorylation of ser122in the amino terminus of ER‐α. Increased phosphorylation was detected within 5 minutes of exposure to estrogen and 5 minutes after the end of the period of strain. Estrogen and strain also activated the mitogen‐activated protein kinase (MAPK) family member extracellular regulated kinase‐1 (ERK‐1). Increases in ERK activation coincided with increased ER‐α phosphorylation. Activation of ERK‐1 and the phosphorylation of ER‐α, by both estrogen and strain, were prevented by the MAP kinase kinase (MEK) inhibitor U0126 and the protein kinase A (PKA) inhibitor (PKI). These data support previous suggestions that resident bone cells' early responses to strain and estrogen share a common pathway, which involves ER‐α. This pathway also appears to involve PKA and ERK‐mediated phosphorylation of ser122within the amino terminus of ER‐α. Reduced availability of this pathway when estrogen levels are reduced could explain diminished effectiveness of mechanically related control of bone architecture after the menopause.