• mechanical strain;
  • osteoblast;
  • estrogen receptor;
  • ERα knockout mouse;
  • mechanotransduction


  1. Top of page
  2. Abstract
  7. Acknowledgements

Osteoblast-like cells in primary cultures derived from ERα−/− mice do not proliferate in response to mechanical strain, unlike those from their ERα+/+ littermates. ERα−/− cells also lack strain-related NO production and responsiveness to IGFs. Proliferative responsiveness to strain is rescued by transfection with functional ERα. ERα number or function in bone cells may limit bones' adaptability to mechanical loading.

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) α and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERα.

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 ERα knockout (ERKO) mice (ERα−/−) with those from their wildtype (ERα+/+) and heterozygous (ERα+/−) littermates and from ERβ knockout (BERKO) mice (ERβ+/+, ERβ+/−, and ERβ−/−).

Results: Whereas ERα+/+, ERα+/−, ERβ+/+, and ERβ−/− cells proliferate in response to a single 10-minute period of cyclic strain, ERα−/− cells do not. Transfection of fully functional, but not mutant, ERα rescues the proliferative response to strain in these cells. The strain-related response of ERα−/− cells is also deficient in that they show no increased activity of an AP-1 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 ERα in the IGF axis and the ability of ERα−/− cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERα.

Conclusions: These data indicate ERα'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 ERα expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.


  1. Top of page
  2. Abstract
  7. Acknowledgements

THE ABILITY OF the skeleton to withstand load-bearing, without damage, is dependent on bone cell populations being able to establish and subsequently maintain structurally appropriate bone architecture. Resident bone cells are commonly believed to do this by adjusting their modeling and remodeling activity in response to the magnitude and distribution of strains that load-bearing engenders within the bone tissue.(1,2) The ability of bone cells to continue to maintain an appropriate match between structure and load-bearing seems to become less effective with estrogen withdrawal at the menopause. The result is reduced bone mass, less structurally effective bone architecture, and increased incidence of fracture. Despite this suggestive link between estrogen and loading-related remodeling, there are no data, to our knowledge, identifying a mechanism by which estrogen itself directly affects bone cells' adaptive response to strain.

In vivo bones' mechanically adaptive response is presumed to involve some or all of the range of osteoblast and osteocyte responses reported to be stimulated by mechanical strain and/or fluid flow in vitro. These include stretch-activated ion channels,(2–5) G-proteins,(6) release of Ca2+ from intracellular stores,(7,8) increases in G6PD activity,(9) increased cyclic adenosine monophosphate (cAMP)(10) and phosphoinositide 3-kinase levels,(11) increased protein kinase C (PKC) activity,(12) MAPK activation,(13–15) ERα phosphorylation,(16) release of NO and prostaglandins,(5,17–19) upregulation of insulin-like growth factor (IGF)-II mRNA,(20) and Cbfa1 expression(21) and proliferation.(22,23)

Previous in vitro work in our laboratory has implicated a role for estrogen receptor α (ERα) in some of these responses to mechanical strain. For example, the strain-related proliferation of primary rat long bone-derived osteoblasts from both males and females is inhibited by tamoxifen, a partial ER antagonist, and by ICI 182,780, a full ER antagonist.(24,25) Exposure of osteoblast-like cells to strain also increases phosphorylation of ERα, in an extracellular regulated kinase (ERK)-dependent manner,(16) and is accompanied by upregulation of estrogen response element (ERE) activity.(26) Moreover, we have previously shown that the proliferative response of ROS 17/2.8 cells to a single period of strain is augmented by transfection of additional ERα.(26) This involvement of ERα in bone cells' adaptive responses to their mechanical environment has led us to hypothesize(27) that the bone loss associated with the menopause is caused by a reduction in bone cell responsiveness to mechanical strain after the reduction in ERα number and/or function associated with estrogen deficiency. Such reductions have been observed previously.(28–30)

ERα is a ligand-inducible transcription factor responsible for mediating the actions of estrogen in target cells.(31,32) It has a modular structure consisting of distinct functional domains including a DNA binding domain, a ligand-binding domain, and two transcriptional activation functions.(33,34) Activation-function 1 (AF-1) is located in the amino-terminal domain and AF-2 in the ligand-binding domain (C terminus). The traditional interpretation of ERα activation involves ligand binding, receptor dimerization, phosphorylation, and interaction with ERE sequences in the promoter region of target genes. There are now, however, numerous examples of both ligand-independent ERα activation(16,32,35–37) and important nongenomic actions.(38–42) ERα has also been shown to influence gene expression through alternative response elements(43–45) and by protein-protein interactions rather than DNA binding.(45–47) Ligand-independent activation of ERα has been shown to occur through both AF-1 and AF-2 domains.(35–37,48)

To address the role of ER, mice with disruption of ERα (ERα knockouts [ERKO]) and ERβ (BERKO) have been generated.(49,50) Longitudinal bone growth is reduced in the female ERKO mice but increased in female BERKO mice, in which there are corresponding changes in serum IGF-I levels.(51–53) Trabecular and cortical BMC are higher in female ERKO mice. However at 12 weeks of age, levels of bone formation and resorption are reduced relative to wildtype (WT) littermates.(53,54) The male ERKO mouse also has shortened long bones and decreased IGF-1 levels in serum.(55,56) Contrary to the situation in females, male ERKO mice have lower BMC compared with WT littermates.(55,56) The mechanical strength of femurs from 17-week-old ERKO males, determined at mid-diaphysis, is lower than in their WT littermates.(56)

If ERα is obligatorily involved in bone's mechanically adaptive responses, ERα knockout animals should have a “disuse” phenotype and should show a defective response to artificial loading. The phenotype of the ERKO mouse is not unambiguously evocative of “disuse,” a fact that may be related to factors such as the high endogenous levels of estrogen. Nevertheless, the ulna in ERKO mice is straighter than in WT littermates (a “disuse” phenomenon),(57) and these bones exhibit a substantially lower osteogenic response to loading in vivo.(58)

This study investigates the mechanisms of ER's involvement in bone cells' adaptive responses to mechanical strain. We therefore compared the response of primary cultures of osteoblast-like cells from ERKO homozygous (ERα−/−), heterozygous (ERα+/−), WT (ERα+/+), and BERKO (ERβ+/+ and ERβ−/−) mice to mechanical strain and IGFs. The responses examined included proliferation, ERK activation, activation of an AP-1 reporter vector derived from the promoter region of the IGF-II gene, and the production of NO and prostaglandins. We also examined the ability of ERα mutants to rescue the deficient responses of ERα−/− cells.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Colony details

The genetic background of both the ERKO and BERKO is the mouse strain C57B16J. The mice were housed in polycarbonate or polypropylene cages with wood chip and paper bedding. Weaners up to 8 weeks of age were fed a standard rodent breeding diet and thereafter a standard rodent maintenance diet (Special Diet Services, South Witham, UK). Postweaning mice were housed in single-sex littermate groups of two or three. They were not segregated by genotype, and no differences in physical activity among the different genotypes were observed. The bone density of these animals was not measured, but this information has been published previously.(52,53,56,59) Ex vivo calibration experiments confirmed that loading engendered the same strain levels in the ulna of knockout and WT mice.(60)

Cell isolation

Osteoblast-like cells were isolated from cortical explants of the long bones of 6- to 10-week-old male and female ERKO and BERKO homozygotes, heterozygotes, and WT littermates by a method described previously.(20) Briefly, this involved the removal of the attendant soft tissue from the long bone and flushing out the bone marrow with sterile PBS. The bones were cut longitudinally, and each half was cut into small (∼1 mm) fragments. The bone fragments were cultured in tissue culture petri dishes (Corning) in DMEM (Invitrogen, Paisley, UK) containing heat-inactivated 10% FCS (Invitrogen), supplemented with 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen) and incubated at 37°C in a humidified 5% CO2 incubator. The fragments were incubated for 21 days to allow cell outgrowth, with media replenished weekly.

Exposure to mitogens

Cells were seeded onto sterile, tissue culture-treated, plastic strips (Nunc, Dossel, Germany) at a density of 80,000 cells/strip, and three strips were incubated together in 150-cm2 plastic sterile dishes (VWR, Poole, UK) with DMEM and 10% charcoal-dextran stripped FCS throughout the treatment period. The cells (three strips of cells/condition) were exposed to mechanical strain. Cells were seeded at a density of 30,000 cells/well (three wells/condition) in 8-well plates (Nunc) and exposed to 2 ng/ml fibroblast growth factor (FGF)-2 or vehicle (media), 40 ng/ml (des 1-3) IGF-I (GroPep, Adelaide, Australia), 40 ng/ml (des 1-6) IGF-II (GroPep), or a vehicle (final concentration, 4 μM HCl) control for 24 h. The IGFs used were truncated to prevent interaction with IGF-binding proteins. The cells were washed in PBS and fixed for 10 minutes in neutral-buffered formalin (BDH), washed twice in PBS, and stored at 4°C.

Mechanical straining

Cells seeded onto plastic strips were subjected to mechanical stimulation in a custom-designed loading apparatus. Each well of the loading apparatus contained one strip and 10 ml of fresh DMEM containing 10% charcoal dextran-stripped media. The strips were subjected to a single 10-minute stimulus of four-point bending consisting of 600 1-Hz cycles of strain generating a peak strain of 3400 με, which is within the physiological range engendered by locomotion in vivo.(61) After the 10-minute period of strain, strips were replaced in the plastic dish and incubated for a further 24 h with media transferred from the wells of the loading apparatus. Static (undisturbed) controls were removed from the incubator, provided with fresh media, and replaced in the incubator for the same 24 h. The cells were washed in PBS and either fixed, for cell counting, lysed for protein, or freeze-thawed with reporter lysis buffer (Promega) for reporter gene analysis, and conditioned media were collected and stored at −20°C.

Cell transfection

Cells for transfection were seeded at a density of 50,000 cells/strip and maintained in DMEM containing 10% FCS throughout the transfection. The cells were transiently transfected with a 3-h incubation with Effectene (Qiagen, Crawley, UK) following the manufacturer's recommendations. The plasmids were used at a concentration of 1 μg DNA/strip. To determine the transfection efficiency, cells were transfected with a lac z expression plasmid, at a concentration of 1 μg DNA/strip. β-galactosidase activity was detected by histochemical staining (lac z staining kit; Promega), and the transfection efficiency was determined by cell counting with phase contrast microscopy.

Plasmids for transfection

pRST7-ER is a human WT ERα expression vector, and pRST7-AF-2 is a human ERα mutant lacking a functional AF-2 domain.(33,62) Both were from D McDonnell (Duke University, Durham, NC, USA). L-543A/L-544A is a leucine to alanine mutation of the full-length mouse ERα sequence and was a gift from R White (Imperial College, London, UK). In this mutant, helix 12 of ERα is permanently in the antagonist-induced conformation that prevents interaction with co-activators.(63) The sequence of the AP-1 Luc reporter vector was derived from the IGF-II promoter and was a gift from A Ward (University of Oxford, Oxford, UK).

Nuclei counting

To determine the number of cells after exposure to strain or mitogens, the nuclei of fixed cells were labeled with a flurochrome (Hoechst 33258; Sigma).(64) The fixed strips were washed with distilled water, blotted dry on tissue, and exposed to 2 ml/strip of 1 mg/ml Hoechst 33258 in TNE (10 mM Tris, 1 mM EDTA, 2M NaCl, pH 7.4) buffer or 1 ml/well and incubated in the dark for 30 minutes. Cells were washed three times in TNE buffer. Excess liquid was blotted off on tissue before adding glycerol and a coverslip (VWR). The stained nuclei were visualized (magnification, ×4) on an ultraviolet microscope (Olympus BH-2), and 16 frames were individually counted per strip. The results are expressed as the average number of nuclei per frame for three strips (or wells) per condition in each experiment.

β-Galactosidase assay

After treatment, the cells were washed in PBS and lysed using 1 ml Reporter Lysis Buffer (Promega, Southampton, UK) per strip. The strips were stored overnight at −20°C and thawed before scraping the lysate into a sterile microcentrifuge tube and vortexed for 15 s. The lysates were stored at −70°C. The β-galactosidase assay (Promega) was performed in 96-well plates (Nunc) using cell lysates and measured on a fluorescence plate reader (Bio-Tek Instruments, Winooski, VT, USA). The results were used to normalize the AP-1 Luc reporter gene activity.

Luciferase assay

Luciferase activity was measured in 20-μl cell lysates in opaque 96-well plates using the Luciferase Assay System from Promega and carried out following the manufacturer's instructions. The samples were measured on a Wallac Victor 1420 multilabel Counter using Wallac 1420 Workstation software version 2 (Wallac Oy, Turku, Finland).

Nitrite assay in conditioned media

Because NO is labile, its production could not be measured directly but was assessed from the concentration of nitrite in the medium. Media samples were stored at −20°C before determination of nitrite concentration by chemiluminescence as described previously.(17,65)

Measurement of prostacyclin concentration in conditioned medium

Once the cells were harvested, conditioned medium was collected and stored (−20°C) before determination of 6-keto-PGF1α (the stable hydrolytic product of PGI2) levels by enzyme immunoassay (Assay Designs, Ann Arbor, MI, USA). None of the compounds that were added to the culture medium interfered with the immunoassays, and the assay was carried out following the manufacturer's instructions.

Statistical analyses

The data were analyzed using the (unpaired) Student t-test and SPSS software (version 10.0). Comparisons were made between the static and strain results within the same genotype. In the transfection experiments, statistical analysis was restricted to the static and strain results for the same vector. Comparisons between genotypes or between different expression vectors were not carried out because of the possibility of difference in expression levels. Significance was determined as p = 0.05 or lower.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Cells derived from both male and female animals were used in this study. No apparent differences were observed in their behavior in vitro. Therefore, the results described represent the combined results of both male and female cells.

ERα−/− osteoblast-like cells do not proliferate in response to strain

Osteoblast-like cells from ERKO WT mice (ERα+/+) as well as those from ERKO heterozygous (ERα+/−) and BERKO WT (ERβ+/+), heterozygous (ERβ+/−), and homozygous (ERβ−/−) mice exhibited significantly increased cell number in response to strain. Cells derived from ERα−/− mice showed no such strain-related response (Fig. 1) instead there was a reduction in cell number compared with the static controls. Cells from ERβ−/− mice showed a greater increase in cell number in response to strain than cells derived from ERβ+/− or ERβ+/+ mice. This suggests that ERβ may exert a negative influence on strain-related responses in osteoblast-like cells.

thumbnail image

Figure FIG. 1.. Proliferation of primary murine osteoblasts in response to mechanical strain. ERα+/+, ERα+/−, and ERα−/− osteoblasts and ERβ+/+, ERβ+/−, and ERβ−/− osteoblasts in DMEM + 10% charcoal dextran-stripped FCS were exposed to a single 10-minute period of strain. The cells were fixed 24 h after the end of the strain stimulus, and the nuclei were stained and counted as described in the Materials and Methods section. The percentage change in cell number after exposure to strain compared with static controls is plotted. Error bars represent SEM, with the following p valuesa0.05,b0.03,c0.05, andd0.02. All genotypes from both ERKO and BERKO animals significantly increased in cell number after exposure to strain compared with static controls, except ERα−/− osteoblasts, which did not increase in cell number after strain (n = 4; all ERKO cells were female; two BERKO experiments were with male cells).

Download figure to PowerPoint

ERα−/− osteoblast-like cells exhibit increased proliferation in response to FGF-2, but not IGFs

IGFs have been suggested to mediate bone cell proliferation in response to strain.(23,66) Consistent with this, osteoblast-like cells derived from ERα+/+ mice showed increased cell number when incubated for 24 h with either 40 ng/ml truncated IGF-I or IGF-II (Fig. 2). In contrast, osteoblast-like cells derived from ERα−/− mice showed no increase in number in response to exogenous IGF-II and only a small, but not statistically significant, increase in response to IGF-I.

thumbnail image

Figure FIG. 2.. Proliferation of primary ERKO-derived osteoblasts in response to exogenous IGF-I, IGF-II, and FGF-2. ERα+/+and ERα−/− osteoblasts in DMEM + 10% charcoal dextran-stripped FCS were exposed to 40 ng/ml IGF-I, IGF-II, or vehicle (HCl) control or 2 ng/ml FGF-2 or vehicle for 24 h. The cells were fixed, and the nuclei were stained and counted as described in the Materials and Methods section. The percentage change in cell number after exposure to the mitogens compared with vehicle is plotted. Error bars represent SEM, with the following p values:a0.01,b0.001, andc0001. ERα−/− osteoblasts failed to proliferate in response to exogenous IGFs but were capable of proliferation similar to WT controls when exposed to FGF-2 (n = 3; two females and one male).

Download figure to PowerPoint

Despite this relative insensitivity of ERα−/− osteoblast-like cells to IGFs, both ERα−/− and ERα+/+ osteoblast-like cells showed a robust and equivalent increase in cell number in response to exogenous FGF-2 (Fig. 2).

All cells derived from BERKO mice, regardless of genotype (ERβ+/+, ERβ+/−, ERβ−/−), showed significant proliferative responses to 40 ng/ml IGF-I and 40 ng/ml IGF-II (data not shown).

ERα−/− osteoblast-like cells produce prostaglandins, but not NO, in response to strain

Osteoblast-like cells release both NO(17,19) and prostaglandins(5,6,18–20) after exposure to strain. Blockade of the production of either NO(67) or prostaglandins(68–71) in vivo have been shown to abrogate the osteogenic response to loading.

In this study, media was collected 10 minutes after the end of exposure to mechanical strain and assayed for nitrite as an indicator of NO release. While osteoblast-like cells derived from ERα+/+ mice increased their medium concentration of nitrite in response to strain, there was no such increase in the case of osteoblast-like cells derived from ERα−/− mice (Fig. 3A). Interestingly, the medium conditioned by ERα−/− osteoblast-like cells contained higher basal levels of nitrite than conditioned media from ERα+/+ cells. Exposure to strain failed to increase this further.

thumbnail image

Figure FIG. 3.. The concentration of nitrite and prostacyclin in media from ERKO-derived cells exposed to mechanical strain. Confluent cells in DMEM + 10% charcoal dextran-stripped FCS were exposed to a single 10-minute period of mechanical strain. (A) Media was collected 10 minutes after the end of the strain stimulus, and the nitrite concentration was determined as described in the Materials and Methods section. The mean concentration from three independent experiments (all female) was plotted, and the SEM is represented by error bars. The nitrite concentration is indicative of the amount of NO released by the cells.aThe concentration of nitrite was increased significantly in media derived from ERα+/+ cells; p = 0.03. In ERα−/− cells, the basal levels of nitrite were higher but were reduced after exposure to strain. (B) Media was collected 24 h after the end of the strain stimulus, and the concentration of 6-keto-PGF1α (the stable hydrolytic product of PGI2) was determined as described in the Materials and Methods section. The mean concentration from three independent experiments (all female) was plotted, and the SEM is represented by error bars. The 6-keto-PGF1α concentration is indicative of the amount of prostacyclin released by the cells. These increases were not statistically significant.

Download figure to PowerPoint

The concentration of 6-keto-PGF1α, the stable metabolite of PGI2, was measured in conditioned media from cells 24 h after exposure to a single period of mechanical strain. We found that the medium concentration of 6-keto-PGF1α was increased in response to the application of strain in media conditioned by either ERα+/+ or ERα−/− osteoblast-like cells (Fig. 3B).

ERα−/− osteoblast-like cells do not exhibit activation of an AP-1 reporter construct in response to strain

The activity of an AP-1 driven reporter vector (measured as luciferase activity normalized for β-galactosidase activity) was increased 8 h after exposure to mechanical strain in osteoblast-like cells derived from ERα+/+ mice (Fig. 4). This increase was not observed in osteoblast-like cells derived from ERα−/− mice. However, the basal level of activation of the AP-1 Luc reporter vector was similar in both genotypes.

thumbnail image

Figure FIG. 4.. Strain-related AP-1 activity in ERKO-derived osteoblasts. ERα+/+ and ERα−/− osteoblasts were transfected with 1 μg AP-1 reporter vector and 0.5 μg lac Z control vector per strip, as described in the Materials and Methods section. Cells were exposed to a single 10-minute period of strain 24 h after transfection and harvested 8 h after the end of the strain stimulus. Activity of the luciferase reporter gene was normalized for β-galactosidase activity. Strain-related increases in AP-1 activity were reduced in ERα−/− compared with ERα+/+ cells (n = 3, all female), but basal levels of activation were similar. These strain-related increases in luciferase activity were not statistically significant.

Download figure to PowerPoint

Transfection with fully functional, but not mutant, ERα confers strain-related proliferation on ERα−/− cells

ERα−/− osteoblast-like cells transfected with fully functional ERα (pRST7-ERα) or with mutant ERα dysfunctional in one or more domains (pRST7-AF-2, L-543A/L-544A) were strained, and cell nuclei counted as described previously. Transfection of fully functional ERα (pRST7-ER) into these cells conferred a proliferative response to strain (Fig. 5), whereas transfection of the AF-2 mutant ERα did not. Transfection of the L-543A/L-544A mutant (in which the helix 12 region is misaligned) produced a small, but not statistically significant increase in cell number following strain compared with the ERα−/− controls. The transfection efficiency is these experiments was measured by histochemical detection of β-galactosidase activity and determined to be 20 ± 2%.

thumbnail image

Figure FIG. 5.. The effect of ERα transfection on strain-related proliferation in ERKO-derived homozygous osteoblasts. ERα−/− osteoblasts in DMEM + 10% FCS were transfected with 1 μg ERα expression vector per strip, as described in the Materials and Methods section. The transfection efficiency was determined to be 20 ± 2%. Cells were exposed to a single 10-minute period of strain in DMEM + 10% charcoal dextran-stripped FCS 24 h after transfection. The cells were fixed 24 h after the end of the strain stimulus, and the nuclei were stained and counted as described in the Materials and Methods section. The percentage change in cell number after exposure to strain compared with static controls is plotted. Error bars represent SEM, with p values as follows:a0.05,b0.004, andc0.02. Transfection of the full-length ERα rescued the strain-related proliferation in ERα−/− cells (n = 5, all female).

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  7. Acknowledgements

The aim of this study was to determine which aspects of ER activity are involved in bone cells' responses to mechanical stimulation. To do this, we examined the responses to mechanical strain and IGFs of primary cultures of long bone-derived osteoblast-like cells from mice with different complements of functional and dysfunctional ERs.

In vivo a consistent and repeatable response to mechanical loading is periosteal and endosteal new bone formation.(66–70) An early feature of this response is proliferation of the cells in the osteogenic layers of both periosteum and endosteum.(69) Without such proliferation, any osteogenic response to load-bearing would be confined to the new bone formation produced by the osteoblasts already active on the bone surface. An inference from the observation that ER antagonists block strain-related proliferation in vitro(24,25) is that strain-related proliferation of osteoblast-like cells requires a functional ER. The data presented here support that inference.

Our current experiments confirm that, when exposed to a short period of dynamic strain, within the physiological strain range, osteoblast-like cells that possess functional ERα (ERα+/+ or ERα+/−) proliferate. In cells where ERα is dysfunctional (ERα−/−) exposure to strain does not stimulate proliferation. This lack of responsiveness can be rescued by transfection of the gene for intact ERα. Because ERα−/− cells contain functional ERβ and do not proliferate with strain, and ERβ−/− cells contain no ERβ but do proliferate with strain, it seems that ERβ is neither normally involved in the proliferative response to strain nor can it substitute for absent or dysfunctional ERα. The finding that strain-related proliferation in cells lacking ERβ is greater than cells with a full complement of ERβ perhaps suggests a negative influence of ERβ on this response. However, we cannot exclude the possibility that the expression of ERα is altered in these cells, which may also account for the increased response.

In addition to their lack of proliferation in response to strain, osteoblast-like cells derived from ERα−/− mice also fail to proliferate in response to exogenous IGF-I and IGF-II. This contrasts with the response of cells from ERα+/+ and ERα+/− littermates. We have shown previously that ROS 17/2.8 osteoblast-like cells show an increase in proliferation in response to estradiol, which can be blocked by neutralizing antibodies to IGF-I, and strain, which can blocked by neutralizing antibodies to IGF-II.(23,72) That osteoblast-like cells from ERα−/− do not proliferate in response to either strain or exogenous IGFs implies that ERα, and not ERβ, is involved in the mitogenic response to both these stimuli. The cell counting assay used in this study does not exclude the possibility that some of the effects we have attributed to proliferation may be contributed to by changes in cell viability.

A number of ERα isoforms have been identified recently, including a 46-kDa truncated ERα isoform expressed in a number of cell types(73,74) and a 55-kDa truncated isoform, resulting specifically from the mutated ERα gene in ERKO-derived cells.(75,76) Both of these isoforms have a truncated A/B domain lacking a functional AF-1 region, but still containing AF-2 and ligand binding domains. The importance of having a full-length, fully functional ERα to mediate bone cells' proliferative response to strain is not only shown by the inability of the truncated forms present in ERKO homozygotes to do so but also by the failure of mutated ERα to rescue this response after transfection. Introduction of the AF-2 mutant, which has a functional AF-1 domain, had no effect on strain-related proliferation, suggesting that a functional AF-1 domain is not sufficient to mediate strain-related proliferation any more than the endogenous AF-2 region of the truncated isoforms. Thus, it is likely that ERα requires both AF-1 and AF-2 regions to mediate a proliferative response to strain.

The L-543A/L-544A mutant has both functional AF-1 and AF-2 regions. Its introduction supported a small increase in strain-related proliferation in ERα−/− osteoblast-like cells, but this was not statistically significant. This mutant contains a misaligned helix 12 region that is known to be involved in receptor interaction with co-activators.(77) Consequently, it seems that strain-related proliferation of osteoblast-like cells requires the presence of both functional AF-1 and AF-2 regions of ERα and, to a lesser extent, an appropriately aligned helix 12 region. However, caution should be taken in comparing the effects of different mutants in case of variation in expression levels. We have shown previously that tamoxifen blocks strain-related proliferation in primary rat long bone-derived osteoblasts.(24) This inhibition would be consistent with tamoxifen blocking AF-2 activity by disruption of helix 12 conformation.(31,33,77,78) This raises an important issue regarding the possible involvement of a ligand in the use of ERα by strain. Tamoxifen induces a receptor conformation that is distinct from that of estrogen.(79) Preliminary results comparing the peptide recruitment of ERα in response to strain and estrogen suggest a distinct ERα conformation induced by strain (D. Ong, personal communication, February 6, 2003). It is likely that the inhibitory effects of tamoxifen observed previously(24) relate to the production of a receptor conformation that inhibits its use by strain rather than inhibition by competition for binding with an endogenous ligand.

It has been reported that female ERKO-derived endothelial cells from the aorta still produce NO in response to estrogen.(80) However, in cerebral arteries of female ERKO mice estrogen-related increases in eNOS and COX-1 were reported to be absent.(75) In this study, ERKO-derived bone cells failed to produce NO in response to mechanical strain, but spontaneous NO release from these cells was enhanced. It is possible that the failure to increase NO production in response to strain is a direct consequence of these high basal levels. The high basal levels may reflect aberrant downstream signaling in ERα−/− osteoblast-like cells, resulting in the loss of negative feedback regulation of NOS activity. The inhibition of NOS in vivo blocks loading-related bone formation(81,82) and the application of NO donors can prevent ovariectomy-induced bone loss,(83) but the signaling cascades that mediate responsiveness to NO are not well-defined in bone. With respect to this study, the significance of the loss of strain-related increases in NO production to the absence of strain-related and IGF-related proliferation has yet to be determined. This is complicated by the observation that, because of the high basal level of NO in ERα−/− osteoblast-like cells, the poststrain levels of nitrite in conditioned media from ERα−/− and ERα+/+ osteoblast-like cells are actually similar although the downstream events differ. This suggests ERα−/− osteoblast-like cells may be relatively insensitive to NO, whether basal or induced.

A number of in vitro studies have shown that bone cells produce prostaglandins in response to mechanical stimulation by strain and/or fluid flow.(5,6,18–20) In vivo blockade of prostaglandin production prevents the osteogenic response to load-bearing.(68–70) Exogenous prostaglandins have been shown to increase the activity of an ERE-driven reporter in osteoblast-like cells,(26) suggesting that the strain-related production of prostaglandins is upstream of ERα-dependent transcription. The current observation that ERα−/− osteoblast-like cells retain strain-related increases in prostaglandin production supports this.

The lack of responsiveness, in terms of strain-related proliferation, may relate to the reduced AP-1 response observed in ERα−/− osteoblast-like cells. ERα is known to function at AP-1 binding sites in the promoter of some target genes, including IGFs,(45,84) and functional ERα AF-1 and AF-2 regions seem to mediate this interaction.(85) The mutated ERα isoforms present in the nucleus of ERα−/− osteoblast-like cells may be unable to carry out this function effectively. This may result in a significant reduction in the production of IGF-II in response to strain. Loss of IGF-II production may explain the lack of strain-related cell proliferation observed in ERα−/− osteoblast-like cells.

The inability of ERα−/− cells to proliferate in response to IGFs is not confined to osteoblast-like cells because uterine cells are similarly unresponsive.(86) Kahlert et al.(87) have demonstrated a physical interaction between ERα and the IGF-I receptor in response to estrogen, resulting in IGF-IR phosphorylation. This interaction is dependent on ERα phosphorylation mediated by ERK. This ERK-mediated phosphorylation occurs in the N-terminal region of ERα, which is truncated in the isoforms expressed in ERKO-derived homozygous cells. Absence of the physical interaction described by Kahlert et al.(87) in ERα−/− cells offers a possible explanation for the lack of proliferation in response to exogenous IGFs.

In summary, our findings support the implication of previous studies that a reduction in ERα expression or activity, or even an alteration in the ERα isoforms present, can abrogate many of osteoblast-like cells' responses to mechanical strain. The responses affected include proliferation, responsiveness to IGFs, NO production, and the activation of ERK and AP-1. Prostaglandin production seems unaffected. Taking into consideration the high levels of estrogen produced by the ERKO mouse, it is evident that it is the activity of the ER and not the presence of estrogen itself that is the determining factor in the ability of bone cells to respond to strain. This inference is supported by the phenotype of a man with an ER null mutation and elevated estrogen levels(88) and the findings of Braidman et al.,(89) who have demonstrated a reduction in ERα expression in young men with idiopathic osteoporosis who are nonetheless estrogen replete. The loss of estrogen after menopause (or ovariectomy) has been shown to result in a decrease in protein and mRNA levels of ERα(29,30) and in ERα activity.(28) This situation, in vivo, would lead to reduced responsiveness to mechanical loading, which, for cells downstream of this response, would be the same as disuse. We hypothesize that the characteristic, functionally inappropriate, bone loss at the menopause reflects the skeleton's remodeling response to apparent disuse that arises at least in part from less effective processing of strain-related information by ERα.


  1. Top of page
  2. Abstract
  7. Acknowledgements

We are grateful to Ken Korach for permission to use the ERKO mouse, to Donald McDonnell and Roger White for providing ER mutant expression vectors and to A. Ward for the AP-1 reporter vector. We also thank the Wellcome Trust & the BBSRC for funding the work.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  • 1
    Lanyon LE 1992 Control of bone architecture by functional load bearing. J Bone Miner Res 7: S2; S369S375.
  • 2
    Duncan RL, Turner CH 1995 Mechanotransduction and the functional response of bone to mechanical strain. Calcif Tissue Int 57: 344358.
  • 3
    Duncan RL, Hruska KA 1994 Chronic, intermittent loading alters mechanosensitive channel characteristics in osteoblast-like cells. Am J Physiol 267: F909F916.
  • 4
    Harter LV, Hruska KA, Duncan RL 1995 Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation. Endocrinology 136: 528535.
  • 5
    Rawlinson SCF, El Haj AJ, Minter SL, Bennet A, Tavares IA, Lanyon LE 1991 Load-related release of prostaglandins in cores of cancellous bone in culture—a role for prostacyclin in adaptive bone remodelling. J Bone Miner Res 6: 13451351.
  • 6
    Reich KM, Mcallister TN, Gudi S, Frangos JA 1997 Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts. Endocrinology 138: 10141018.
  • 7
    Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, Duncan RL 2000 Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol 278: C989C997.
  • 8
    Donahue SW, Donahue HJ, Jacobs CR 2003 Osteoblastic cells have refractory periods for fluid-flow-induced intracellular calcium oscillations for short bouts of flow and display multiple low-magnitude oscillations during long-term flow. J Biomech 36: 3543.
  • 9
    Dodds RA, Ali N, Pead MJ, Lanyon LE 1993 Early loading related changes in the activity of glucose 6-phosphate dehydrogenase and alkaline phosphatase in osteocytes and periosteal osteoblasts in rat fibulae in vivo. J Bone Miner Res 8: 261267.
  • 10
    Mikuni-Takagaki Y 1999 Mechanical responses and signal transduction pathways in stretched osteocytes. J Bone Miner Metab 17: 5760.
  • 11
    Carvalho RS, Scott JE, Suga DM, Yen EHK 1994 Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone. J Bone Miner Res 9: 9991011.
  • 12
    Geng WD, Boskovic G, Fultz ME, Li C, Niles RM, Ohno S, Wright G 2001 Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells. J Cell Physiol 189: 216228.
  • 13
    Seko Y, Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y 1999 Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125 FAK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun 259: 814.
  • 14
    Peverali FA, Basdra EK, Papavassiliou AG 2001 Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells. Mol Med 7: 6878.
  • 15
    Jessop HL, Rawlinson SCF, Pitsillides AA, Lanyon LE 2002 Mechanical strain and fluid movement both activate ERK in osteoblast-like cells but via different signalling pathways. Bone 31: 186194.
  • 16
    Jessop HL, Sjöberg M, Cheng MZ, Zaman G, Wheeler-Jones CPD, Lanyon LE 2001 Mechanical strain as well as estrogen activates estrogen receptor α in bone cells. J Bone Miner Res 16: 10451055.
  • 17
    Pitsillides AA, Rawlinson SCF, Suswillo RFL, Bourrin S, Zaman G, Lanyon LE 1995 Mechanical strain-induced NO production by bone cells: A possible role in adaptive bone (re)modelling? FASEB J 9: 16141622.
  • 18
    Ajubi NE, Klein-Nulend J, Alblas MJ, Burger EH, Nijweide PJ 1999 Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes. Am J Physiol 276: E171E178.
  • 19
    Klein-Nulend J, Semeins CM, Ajubi NE, Nijweide PJ, Burger EH 1995 Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts: Correlation with prostaglandin upregulation. Biochem Biophys Res Commun 217: 640648.
  • 20
    Zaman G, Suswillo RFL, Cheng MZ, Tavares IA, Lanyon LE 1997 Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture. J Bone Miner Res 12: 769777.
  • 21
    Ziros PG, Gil A-PR, Georgakopoulos T, Habeos I, Kletsas D, Basdra EK, Papavassiliou AG 2002 The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells. J Biol Chem 277: 23934239341.
  • 22
    Zhuang H, Wang W, Tahernia AD, Levitz CL, Luchetti WT, Brighton CT 1996 Mechanical strain-induced proliferation of osteoblastic cells parallels increased TGF-1β mRNA. Biochem Biophys Res Commun 229: 449453.
  • 23
    Cheng MZ, Zaman G, Rawlinson SCF, Mohan S, Baylink DJ, Lanyon LE 1999 Mechanical strain stimulates ROS cells proliferation through IGF-II and estrogen through IGF-I. J Bone Miner Res 10: 17421750.
  • 24
    Damien E, Price JS, Lanyon LE 1998 The estrogen receptor's involvement in osteoblasts adaptive response to mechanical strain. J Bone Miner Res 13: 12751282.
  • 25
    Damien E, Price JS, Lanyon LE 2000 Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females. J Bone Miner Res 15: 21692177.
  • 26
    Zaman G, Cheng MZ, Jessop HL, White R, Lanyon LE 2000 Mechanical strain activates estrogen response elements in bone cells. Bone 27: 233239.
  • 27
    Lanyon LE, Skerry T 2001 Postmenopausal osteoporosis as a failure of bone's adaptation to functional loading: A hypothesis. J Bone Miner Res 16: 19371947.
  • 28
    Ankrom MA, Patterson JA, d'Avis PY, Vetter UK, Blackman MR, Sponsellers PD, Tayback M, Robey PG, Shapiro JR, Fedarko NS 1998 Age-related changes in human oestrogen receptor α function and levels in osteoblasts. Biochem J 333: 787794.
  • 29
    Hoyland JA, Baris C, Wood L, Baird P, Selby PL, Freemont AJ, Braidman IP 1999 Effect of ovarian steroid deficiency on oestrogen receptor α expression in bone. J Pathol 188: 294303.
  • 30
    Lim SK, Won YJ, Lee HC, Huh KB, Park YS 1999 A PCR analysis of ERalpha and ER beta mRNA abundance in rats and the effect of ovariectomy. J Bone Miner Res 14: 11891196.
  • 31
    Parker MG, Arbuckle N, Dauvois S, Danielian P, White R 1993 Structure and function of the estrogen receptor. Ann NY Acad Sci 684: 119126.
  • 32
    Smith CL 1998 Cross-talk between peptide growth factor and estrogen receptor signalling pathways. Biol Reprod 58: 627632.
  • 33
    McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 9: 659669.
  • 34
    Norris JD, Fan D, Stallcup MR, McDonnell DP 1998 Enhancement of estrogen receptor transcriptional activity by the co-activator GRIP-1 highlights the role of AF-2 in determining estrogen receptor pharmacology. J Biol Chem 273: 66796688.
  • 35
    Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated-protein kinase. Science 270: 14911494.
  • 36
    Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15: 21742183.
  • 37
    El-Tanani MKK, Green CD 1997 Two separate mechanisms for ligand-independent activation of the estrogen receptor. Mol Endocrinol 11: 928937.
  • 38
    Sömjen D, Kohen F, Lieberherr M 1997 Nongenomic effects of an anti-idiotypic antibody as an estrogen mimetic in female human and rat osteoblasts. J Cell Biochem 65: 5366.
  • 39
    Nadal A, Ropero AB, Laribi O, Maillet M, Fuentes E, Soria B 2000 Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to ERα and ERβ. Proc Natl Acad Sci USA 97: 1160311608.
  • 40
    Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407: 538541.
  • 41
    Kousteni S, Bellido T, Plotkin LI, O'Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, Manolagas SC 2001 Non-genotropic, sex non-specific signalling through the estrogen or androgen receptors: Dissociation from transcriptional activity. Cell 104: 719730.
  • 42
    Wong C-W, McNally C, Nickbarg E, Komm BS, Cheskis BJ 2002 Estrogen receptor-interacting protein that modulates its nongenomic activity with Src/ERK phosphorylation cascade. Proc Natl Acad Sci USA 99: 1478314788.
  • 43
    Li C, Briggs MR, Ahlborn TE, Kraemer FB, Liu J 2001 Requirement of Sp1 and estrogen receptor α interaction in 17β-estradiol-mediated transcriptional activation of low density lipoprotein receptor gene expression. Endocrinology 142: 15461553.
  • 44
    Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ERα and ERβ at AP-1 sites. Science 277: 15081510.
  • 45
    Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/ AP-1 pathway: Potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9: 443456.
  • 46
    Gaub MP, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P 1990 Activation of ovabumine gene by the estrogen receptor involves the Fos-Jun complex. Cell 63: 12671276.
  • 47
    Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11: 15691580.
  • 48
    Ali S, Metzger D, Bornert J-M, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B domain. EMBO J 12: 11531160.
  • 49
    Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90: 1116211166.
  • 50
    Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor-β. Proc Natl Acad Sci USA 95: 1567715682.
  • 51
    Couse JF, Korach KS 1999 Estrogen receptor null mice: What have we learned and where will they lead us? Endocr Rev 20: 358417.
  • 52
    Vidal O, Lindberg MK, Savendahl L, Lubahn DB, Ritzen EM, Gustafsson JA, Ohlsson C 1999 Disproportional body growth in female estrogen receptor-alpha-inactivated mice. Biochem Biophys res Commun 265: 569571.
  • 53
    Lindberg MK, Alatalo SL, Halleen JM, Mohan S, Gustafsson JA, Ohlsson C 2001 Estrogen receptor specificity in the regulation of the skeleton in female mice. J Endocrinol 171: 229236.
  • 54
    Sims NA, Clement-Lacroix P, Minet D, Dupont S, Krust A, Resche-Rigon M, Gaillard-Kelly M, Chambon P, Baron R 2001 Estrogen receptor α is the major receptor regulating bone response to estradiol in gonadectomised female mice and testosterone plays a role in intact male and female ER α knockout mice. J Bone Miner Res 16: S431.
  • 55
    Vandenput L, Edervenn AG, Erben RG, Stahr K, Swinnen JV, Van Herck E, Verstuyf A, Boonen S, Bouillon R, Vanderschueren D 2001 Testosterone prevents orchidectomy-induced bone loss in estrogen receptor alpha knockout mice. Biochem Biophys Res Commun 285: 7076.
  • 56
    Vidal O, Lindberg MK, Hollberg K, Baylink DJ, Andersson G, Lubahn DB, Mohan S, Gustafsson JA, Ohlsson C 2000 Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice. Proc Natl Acad Sci USA 97: 54745479.
  • 57
    Lanyon LE 1974 Experimental support for the trajectorial theory of bone structure. J Bone Joint Surg Br 56: 160166.
  • 58
    Lee K, Jessop H, Suswillo R, Zaman G, Lanyon L 2003 Bone adaptation requires oestrogen receptor-α. Nature 424: 389.
  • 59
    Windahl SH, Hollberg K, Vidal O, Gustafsson JA, Ohlsson C, Andersson G 2001 Female estrogen receptor beta -/- mice are partially protected against age-related trabecular bone loss. J Bone Miner Res 16: 13881398.
  • 60
    Lee KCL 2003 Structural adaptation of the mouse ulna to mechanical loading and the involvement of estrogen receptor alpha. Ph.D. thesis, University of London, London, UK.
  • 61
    Rubin CT, Lanyon LE 1984 Dynamic strain similarity in vertebrates: An alternative to allometric limb bone scaling. J Theor Biol 107: 321327.
  • 62
    Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transcriptional capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8: 2130.
  • 63
    Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 11: 10251033.
  • 64
    Rago R, Mitchen J, Wilding G 1990 DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191: 3134.
  • 65
    Palmer RM, Ferrigo AG, Moncada S 1987 Nitric oxide release accounts for biological activity of endothelium-derived releasing factor. Nature 325: 524525.
  • 66
    Jagger CJ, Chow JWM, Chambers TJ 1996 Estrogen suppresses activation but enhances formation phase of osteogenic response to mechanical stimulation in rat bone. J Clin Invest 98: 23512357.
  • 67
    Turner CH, Takano Y, Owan I, Murrell GAC 1996 Nitric oxide inhibitor L-NAME suppresses mechanically induced bone formation in rats. Am J Physiol 270: E634E639.
  • 68
    Tornkvist H, Lindholm TS, Netz P, Stromberg L, Lindholm TC 1984 Effect of Ibuprofen and indomethacin on bone metabolism reflected in bone strength. Clin Orthop 187: 255259.
  • 69
    Pead MJ, Lanyon LE 1989 Indomethacin modulation of load-related stimulation of new bone formation in vivo. Calcif Tissue Int 45: 3440.
  • 70
    Keller J, Bayer-Kristensen I, Bak B, Bunger C, Kjaersgaard-Andersen P 1989 Indomethacin and bone remodelling. Effect on cortical bone after osteotomy in rabbits. Acta Orthop Scand 60: 119121.
  • 71
    Chambers TJ, Fox S, Jagger CJ, Lean JM, Chow JWN 1999 The role of prostaglandins and nitric oxide in the response of bone to mechanical forces. Osteoarthritis Cartilage 7: 422423.
  • 72
    Cheng MZ, Rawlinson SCF, Pitsillides AA, Zaman G, Mohan S, Baylink DJ, Lanyon LE 2002 Human osteoblasts' proliferative responses to strain and 17beta-estradiol are mediated by the estrogen receptor and the receptor for insulin-like growth factor I. J Bone Miner Res 17: 593602.
  • 73
    Resnick EM, Schreihofer DA, Periasamy A, Shupnik MA 2000 Truncated estrogen receptor product-1 suppresses estrogen receptor transactivation by dimerization with estrogen receptors α and β. J Biol Chem 275: 71587166.
  • 74
    Yang J, Liu A, Chiou S-K, Guzman R, Nandi S 2000 Estrogen receptor variants are present in many normal human tissues. Int J Mol Med 5: 223227.
  • 75
    Geary GG, McNeill AM, Ospina JA, Krause DN, Korach KS, Duckles SP 2001 Cerebrovascular NOS and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-α. J Appl Physiol 91: 23912399.
  • 76
    Denger S, Reid G, Koš M, Flouriot G, Parsch D, Brand H, Korach KS, Sonntag-Buck V, Gannon F 2001 ERα gene expression in human primary osteoblasts: Evidence for the expression of two receptor proteins. Mol Endocrinol 15: 20642077.
  • 77
    Berry M, Metzger D, Chambon P 1990 Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9: 28112818.
  • 78
    Willson TM, Norris JD, Wagner BL, Asplin I, Baer P, Brown HR, Jones SA, Henke B, Sauls H, Wolfe S, Morris DC, McDonnell DP 1997 Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone. Endocrinology 138: 39013911.
  • 79
    Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95: 927937.
  • 80
    Pendaries C, Darblade B, Rochaix P, Krust A, Chambon P, Korach KS, Bayard F, Arnal J-F 2001 The AF-1 activation function may be dispensable to mediate the effect of estradiol on endothelial NO production in mice. Proc Natl Acad Sci USA 99: 22052210.
  • 81
    Fox SW, Chambers TJ, Chow JWM 1996 Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation. Am J Physiol 270: E955E960.
  • 82
    Turner CH, Takano Y, Owan I, Murrell GA 1996 Nitric oxide inhibitor L-NAME suppresses mechanically induced bone formation in rats. Am J Physiol 270: E634E639.
  • 83
    Wimalawansa SJ, De Marco G, Gangula P, Yallampalli C 1996 Nitric oxide donor alleviates ovariectomy-induced bone loss. Bone 18: 301304.
  • 84
    Jakacka M, Ito M, Weiss J, Chien P-Y, Gehm BD, Jameson JL 2001 Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP-1 pathway. J Biol Chem 276: 1361513621.
  • 85
    Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P 2000 Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74: 311317.
  • 86
    Klotz DM, Curtis Hewitt S, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS 2002 Requirement of estrogen receptor α in insulin-like growth factor-I (IGF-I)-induced uterine responses and in vivo evidence for IGF-I/estrogen receptor cross-talk. J Biol Chem 277: 85318537.
  • 87
    Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, Grohé C 2000 Estrogen receptor α rapidly activates the IGF-I receptor pathway. J Biol Chem 275: 1844718453.
  • 88
    Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen receptor gene in a man. N Engl J Med 331: 10561061.
  • 89
    Braidman I, Baris C, Wood L, Selby P, Adams J, Freemont A, Hoyland J 2000 Preliminary evidence for impaired estrogen receptor-alpha protein expression in osteoblasts and osteocytes from men with idiopathic osteoporosis. Bone 26: 423427.