The development of veterinary medicine.

Reviewed by Lance Lanyon, former principal of the Royal Veterinary College.

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

Parathyroid hormone's enhancement of bones' osteogenic response to loading is affected by ageing in a dose- and time-dependent manner.

Decreased effectiveness of bones' adaptive response to mechanical loading contributes to age-related bone loss. In young mice, intermittent administration of parathyroid hormone (iPTH) at 20-80µg/kg/day interacts synergistically with artificially applied loading to increase bone mass. Here we report investigations on the effect of different doses and duration of iPTH treatment on mice whose osteogenic response to artificial loading is impaired by age. One group of aged, 19-month-old female C57BL/6 mice was given 0, 25, 50 or 100µg/kg/day iPTH for 4weeks. Histological and µCT analysis of their tibiae revealed potent iPTH dose-related increases in periosteally-enclosed area, cortical area and porosity with decreased cortical thickness. There was practically no effect on trabecular bone. Another group was given a submaximal dose of 50µg/kg/day iPTH or vehicle for 2 or 6weeks with loading of their right tibia three times per week for the final 2weeks. In the trabecular bone of these mice the loading-related increase in BV/TV was abrogated by iPTH primarily by reduction of the increase in trabecular number. In their cortical bone, iPTH treatment time-dependently increased cortical porosity. Loading partially reduced this effect. The osteogenic effects of iPTH and loading on periosteally-enclosed area and cortical area were additive but not synergistic. Thus in aged, unlike young mice, iPTH and loading appear to have separate effects. iPTH alone causes a marked increase in cortical porosity which loading reduces. Both iPTH and loading have positive effects on cortical periosteal bone formation but these are additive rather than synergistic.

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes.

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load-bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18ß-glycyrrhetinic acid, we also demonstrated that this osteocyte-related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial-derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain-related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.

Old age and the associated impairment of bones' adaptation to loading are associated with transcriptomic changes in cellular metabolism, cell-matrix interactions and the cell cycle.

In old animals, bone's ability to adapt its mass and architecture to functional load-bearing requirements is diminished, resulting in bone loss characteristic of osteoporosis. Here we investigate transcriptomic changes associated with this impaired adaptive response. Young adult (19-week-old) and aged (19-month-old) female mice were subjected to unilateral axial tibial loading and their cortical shells harvested for microarray analysis between 1h and 24h following loading (36 mice per age group, 6 mice per loading group at 6 time points). In non-loaded aged bones, down-regulated genes are enriched for MAPK, Wnt and cell cycle components, including E2F1. E2F1 is the transcription factor most closely associated with genes down-regulated by ageing and is down-regulated at the protein level in osteocytes. Genes up-regulated in aged bone are enriched for carbohydrate metabolism, TNFα and TGFß superfamily components. Loading stimulates rapid and sustained transcriptional responses in both age groups. However, genes related to proliferation are predominantly up-regulated in the young and down-regulated in the aged following loading, whereas those implicated in bioenergetics are down-regulated in the young and up-regulated in the aged. Networks of inter-related transcription factors regulated by E2F1 are loading-responsive in both age groups. Loading regulates genes involved in similar signalling cascades in both age groups, but these responses are more sustained in the young than aged. From this we conclude that cells in aged bone retain the capability to sense and transduce loading-related stimuli, but their ability to translate acute responses into functionally relevant outcomes is diminished.

Sclerostin's role in bone's adaptive response to mechanical loading.

Mechanical loading is the primary functional determinant of bone mass and architecture, and osteocytes play a key role in translating mechanical signals into (re)modelling responses. Although the precise mechanisms remain unclear, Wnt signalling pathway components, and the anti-osteogenic canonical Wnt inhibitor Sost/sclerostin in particular, play an important role in regulating bone's adaptive response to loading. Increases in loading-engendered strains down-regulate osteocyte sclerostin expression, whereas reduced strains, as in disuse, are associated with increased sclerostin production and bone loss. However, while sclerostin up-regulation appears to be necessary for the loss of bone with disuse, the role of sclerostin in the osteogenic response to loading is more complex. While mice unable to down-regulate sclerostin do not gain bone with loading, Sost knockout mice have an enhanced osteogenic response to loading. The molecular mechanisms by which osteocytes sense and transduce loading-related stimuli into changes in sclerostin expression remain unclear but include several, potentially interlinked, signalling cascades involving periostin/integrin, prostaglandin, estrogen receptor, calcium/NO and Igf signalling. Deciphering the mechanisms by which changes in the mechanical environment regulate sclerostin production may lead to the development of therapeutic strategies that can reverse the skeletal structural deterioration characteristic of disuse and age-related osteoporosis and enhance bones' functional adaptation to loading. By enhancing the osteogenic potential of the context in which individual therapies such as sclerostin antibodies act it may become possible to both prevent and reverse the age-related skeletal structural deterioration characteristic of osteoporosis.

Wnt16 Is Associated with Age-Related Bone Loss and Estrogen Withdrawal in Murine Bone.

Genome Wide Association Studies suggest that Wnt16 is an important contributor to the mechanisms controlling bone mineral density, cortical thickness, bone strength and ultimately fracture risk. Wnt16 acts on osteoblasts and osteoclasts and, in cortical bone, is predominantly derived from osteoblasts. This led us to hypothesize that low bone mass would be associated with low levels of Wnt16 expression and that Wnt16 expression would be increased by anabolic factors, including mechanical loading. We therefore investigated Wnt16 expression in the context of ageing, mechanical loading and unloading, estrogen deficiency and replacement, and estrogen receptor α (ERα) depletion. Quantitative real time PCR showed that Wnt16 mRNA expression was lower in cortical bone and marrow of aged compared to young female mice. Neither increased nor decreased (by disuse) mechanical loading altered Wnt16 expression in young female mice, although Wnt16 expression was decreased following ovariectomy. Both 17ß-estradiol and the Selective Estrogen Receptor Modulator Tamoxifen increased Wnt16 expression relative to ovariectomy. Wnt16 and ERß expression were increased in female ERα-/- mice when compared to Wild Type. We also addressed potential effects of gender on Wnt16 expression and while the expression was lower in the cortical bone of aged males as in females, it was higher in male bone marrow of aged mice compared to young. In the kidney, which we used as a non-bone reference tissue, Wnt16 expression was unaffected by age in either males or females. In summary, age, and its associated bone loss, is associated with low levels of Wnt16 expression whereas bone loss associated with disuse has no effect on Wnt16 expression. In the artificially loaded mouse tibia we observed no loading-related up-regulation of Wnt16 expression but provide evidence that its expression is influenced by estrogen receptor signaling. These findings suggest that while Wnt16 is not an obligatory contributor to regulation of bone mass per se, it potentially plays a role in influencing pathways associated with regulation of bone mass during ageing and estrogen withdrawal.

Exercise does not enhance aged bone's impaired response to artificial loading in C57Bl/6 mice.

Bones adapt their structure to their loading environment and so ensure that they become, and are maintained, sufficiently strong to withstand the loads to which they are habituated. The effectiveness of this process declines with age and bones become fragile fracturing with less force. This effect in humans also occurs in mice which experience age-related bone loss and reduced adaptation to loading. Exercise engenders many systemic and local muscular physiological responses as well as engendering local bone strain. To investigate whether these physiological responses influence bones' adaptive responses to mechanical strain we examined whether a period of treadmill exercise influenced the adaptive response to an associated period of artificial loading in young adult (17-week) and old (19-month) mice. After treadmill acclimatization, mice were exercised for 30 min three times per week for two weeks. Three hours after each exercise period, right tibiae were subjected to 40 cycles of non-invasive axial loading engendering peak strain of 2250 µÎµ. In both young and aged mice exercise increased cross-sectional muscle area and serum sclerostin concentration. In young mice it also increased serum IGF1. Exercise did not affect bone's adaptation to loading in any measured parameter in young or aged bone. These data demonstrate that a level of exercise sufficient to cause systemic changes in serum, and adaptive changes in local musculature, has no effect on bone's response to loading 3h later. This study provides no support for the beneficial effects of exercise on bone in the elderly being mediated by systemic or local muscle-derived effects rather than local adaptation to altered mechanical strain.

Quantification of Alterations in Cortical Bone Geometry Using Site Specificity Software in Mouse models of Aging and the Responses to Ovariectomy and Altered Loading.

Investigations into the effect of (re)modeling stimuli on cortical bone in rodents normally rely on analysis of changes in bone mass and architecture at a narrow cross-sectional site. However, it is well established that the effects of axial loading produce site-specific changes throughout bones' structure. Non-mechanical influences (e.g., hormones) can be additional to or oppose locally controlled adaptive responses and may have more generalized effects. Tools currently available to study site-specific cortical bone adaptation are limited. Here, we applied novel site specificity software to measure bone mass and architecture at each 1% site along the length of the mouse tibia from standard micro-computed tomography (µCT) images. Resulting measures are directly comparable to those obtained through µCT analysis (R (2) > 0.96). Site Specificity analysis was used to compare a number of parameters in tibiae from young adult (19-week-old) versus aged (19-month-old) mice; ovariectomized and entire mice; limbs subjected to short periods of axial loading or disuse induced by sciatic neurectomy. Age was associated with uniformly reduced cortical thickness and site-specific decreases in cortical area most apparent in the proximal tibia. Mechanical loading site-specifically increased cortical area and thickness in the proximal tibia. Disuse uniformly decreased cortical thickness and decreased cortical area in the proximal tibia. Ovariectomy uniformly reduced cortical area without altering cortical thickness. Differences in polar moment of inertia between experimental groups were only observed in the proximal tibia. Aging and ovariectomy also altered eccentricity in the distal tibia. In summary, site specificity analysis provides a valuable tool for measuring changes in cortical bone mass and architecture along the entire length of a bone. Changes in the (re)modeling response determined at a single site may not reflect the response at different locations within the same bone.

Planar cell polarity aligns osteoblast division in response to substrate strain.

Exposure of bone to dynamic strain increases the rate of division of osteoblasts and also influences the directional organization of the cellular and molecular structure of the bone tissue that they produce. Here, we report that brief exposure to dynamic substrate strain (sufficient to rapidly stimulate cell division) influences the orientation of osteoblastic cell division. The initial proliferative response to strain involves canonical Wnt signaling and can be blocked by sclerostin. However, the strain-related orientation of cell division is independently influenced through the noncanonical Wnt/planar cell polarity (PCP) pathway. Blockade of Rho-associated coiled kinase (ROCK), a component of the PCP pathway, prevents strain-related orientation of division in osteoblast-like Saos-2 cells. Heterozygous loop-tail mutation of the core PCP component van Gogh-like 2 (Vangl2) in mouse osteoblasts impairs the orientation of division in response to strain. Examination of bones from Vangl2 loop-tail heterozygous mice by µCT and scanning electron microscopy reveals altered bone architecture and disorganized bone-forming surfaces. Hence, in addition to the well-accepted role of PCP involvement in response to developmental cues during skeletal morphogenesis, our data reveal that this pathway also acts postnatally, in parallel with canonical Wnt signaling, to transduce biomechanical cues into skeletal adaptive responses. The simultaneous and independent actions of these two pathways appear to influence both the rate and orientation of osteoblast division, thus fine-tuning bone architecture to meet the structural demands of functional loading.

The Contribution of Experimental in vivo Models to Understanding the Mechanisms of Adaptation to Mechanical Loading in Bone.

Changing loading regimens by natural means such as exercise, with or without interference such as osteotomy, has provided useful information on the structure:function relationship in bone tissue. However, the greatest precision in defining those aspects of the overall strain environment that influence modeling and remodeling behavior has been achieved by relating quantified changes in bone architecture to quantified changes in bones' strain environment produced by direct, controlled artificial bone loading. Jiri Hert introduced the technique of artificial loading of bones in vivo with external devices in the 1960s using an electromechanical device to load rabbit tibiae through transfixing stainless steel pins. Quantifying natural bone strains during locomotion by attaching electrical resistance strain gages to bone surfaces was introduced by Lanyon, also in the 1960s. These studies in a variety of bones in a number of species demonstrated remarkable uniformity in the peak strains and maximum strain rates experienced. Experiments combining strain gage instrumentation with artificial loading in sheep, pigs, roosters, turkeys, rats, and mice has yielded significant insight into the control of strain-related adaptive (re)modeling. This diversity of approach has been largely superseded by non-invasive transcutaneous loading in rats and mice, which is now the model of choice for many studies. Together such studies have demonstrated that over the physiological strain range, bone's mechanically adaptive processes are responsive to dynamic but not static strains; the size and nature of the adaptive response controlling bone mass is linearly related to the peak loads encountered; the strain-related response is preferentially sensitive to high strain rates and unresponsive to static ones; is most responsive to unusual strain distributions; is maximized by remarkably few strain cycles, and that these are most effective when interrupted by short periods of rest between them.

Protein kinase Cα (PKCα) regulates bone architecture and osteoblast activity.

Bones' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca(-/-) female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca(-/-) but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca(-/-) mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca(-/-) mice do not. Female Prkca(-/-) mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca(-/-) mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions.

Age-related impairment of bones' adaptive response to loading in mice is associated with sex-related deficiencies in osteoblasts but no change in osteocytes.

Bones adjust their mass and architecture to be sufficiently robust to withstand functional loading by adapting to their strain environment. This mechanism appears less effective with age, resulting in low bone mass. In male and female young adult (17-week-old) and old (19-month-old) mice, we investigated the effect of age in vivo on bones' adaptive response to loading and in vitro in primary cultures of osteoblast-like cells derived from bone cortices. Right tibias were axially loaded on alternate days for 2 weeks. Left tibias were non-loaded controls. In a separate group, the number of sclerostin-positive osteocytes and the number of periosteal osteoblasts were analyzed 24 hours after a single loading episode. The responses to strain of the primary osteoblast-like cells derived from these mice were assessed by EGR2 expression, change in cell number and Ki67 immunofluorescence. In young male and female mice, loading increased trabecular thickness and the number of trabecular connections. Increase in the number of trabecular connections was impaired with age but trabecular thickness was not. In old mice, the loading-related increase in periosteal apposition of the cortex was less than in young ones. Age was associated with a lesser loading-related increase in osteoblast number on the periosteal surface but had no effect on loading-related reduction in the number of sclerostin-positive osteocytes. In vitro, strain-related proliferation of osteoblast-like cells was lower in cells from old than young mice. Cells from aged female mice demonstrated normal entry into the cell cycle but subsequently arrested in G2 phase, reducing strain-related increases in cell number. Thus, in both male and female mice, loading-related adaptive responses are impaired with age. This impairment is different in females and males. The deficit appears to occur in osteoblasts' proliferative responses to strain rather than earlier strain-related responses in the osteocytes.

Evidence-based veterinary medicine: a clear and present challenge.

If evidence-based veterinary medicine is to become a reality, the veterinary profession must be both prepared and in a position to undertake the necessary research, says Lance Lanyon.

Estrogen receptor α mediates proliferation of osteoblastic cells stimulated by estrogen and mechanical strain, but their acute down-regulation of the Wnt antagonist Sost is mediated by estrogen receptor ß.

Mechanical strain and estrogens both stimulate osteoblast proliferation through estrogen receptor (ER)-mediated effects, and both down-regulate the Wnt antagonist Sost/sclerostin. Here, we investigate the differential effects of ERα and -ß in these processes in mouse long bone-derived osteoblastic cells and human Saos-2 cells. Recruitment to the cell cycle following strain or 17ß-estradiol occurs within 30 min, as determined by Ki-67 staining, and is prevented by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride. ERß inhibition with 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-ß]pyrimidin-3-yl] phenol (PTHPP) increases basal proliferation similarly to strain or estradiol. Both strain and estradiol down-regulate Sost expression, as does in vitro inhibition or in vivo deletion of ERα. The ERß agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and ERB041 also down-regulated Sost expression in vitro, whereas the ERα agonist 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl]tris-phenol or the ERß antagonist PTHPP has no effect. Tamoxifen, a nongenomic ERß agonist, down-regulates Sost expression in vitro and in bones in vivo. Inhibition of both ERs with fulvestrant or selective antagonism of ERß, but not ERα, prevents Sost down-regulation by strain or estradiol. Sost down-regulation by strain or ERß activation is prevented by MEK/ERK blockade. Exogenous sclerostin has no effect on estradiol-induced proliferation but prevents that following strain. Thus, in osteoblastic cells the acute proliferative effects of both estradiol and strain are ERα-mediated. Basal Sost down-regulation follows decreased activity of ERα and increased activity of ERß. Sost down-regulation by strain or increased estrogens is mediated by ERß, not ERα. ER-targeting therapy may facilitate structurally appropriate bone formation by enhancing the distinct ligand-independent, strain-related contributions to proliferation of both ERα and ERß.

Male mice housed in groups engage in frequent fighting and show a lower response to additional bone loading than females or individually housed males that do not fight.

Experiments to investigate bone's physiological adaptation to mechanical loading frequently employ models that apply dynamic loads to bones in vivo and assess the changes in mass and architecture that result. It is axiomatic that bones will only show an adaptive response if the applied artificial loading environment differs in a significant way from that to which the bones have been habituated by normal functional loading. It is generally assumed that this normal loading is similar between experimental groups. In the study reported here we found that this was not always the case. Male and female 17-week-old C57BL/6 mice were housed in groups of six, and a single episode (40 cycles) of non-invasive axial loading, engendering 2,200 µÎµ on the medial surface of the proximal tibiae in sample mice, was applied to right tibiae on alternate days for two weeks. This engendered an adaptive increase in bone mass in females, but not males. Observation revealed the main difference in behaviour between males and females was that males were involved in fights 1.3 times per hour, whereas the females never fought. We therefore housed all mice individually. In females, there was a similar significant osteogenic response to loading in cortical and trabecular bone of both grouped and individual mice. In contrast, in males, adaptive increases in the loaded compared with non-loaded control bones was only apparent in animals housed individually. Our interpretation of these findings is that the frequent vigorous fighting that occurs between young adult males housed in groups could be sufficient to engender peak strains and strain rates that equal or exceed the stimulus derived from artificial loading. This indicates the importance of ensuring that physical activity is consistent between groups. Reducing the background level of the naturally engendered strain environment allows adaptive responses to artificial loading to be demonstrated at lower loads.

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-1(0) ), or (3) specific inactivation of ERαAF-2 (ERαAF-2(0) ) 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-1(0) 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-2(0) 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.

Estrogen receptors' roles in the control of mechanically adaptive bone (re)modeling.

The discovery that estrogen receptors (ERs) are involved in bone cells' responses to mechanical strain offered the prospect of establishing the link between declining levels of circulating estrogen and the progressive failure of the mechanically adaptive mechanisms that should maintain structurally appropriate levels of bone mass in age-related and post-menopausal osteoporosis. Such clarification remains elusive but studies have confirmed ligand-independent involvement of ERs as facilitators in a number of the pathways by which mechanical strain stimulates osteoblast proliferation and bone formation. The presence of α and ß forms of ER that oppose, supplement or replace one another has complicated interpretation of studies to identify their individual roles when both are present in normal amounts. However, it appears that, in mice at least, ERα promotes cortical bone mass in both males and females through its effects in early members of the osteoblast lineage, but enhances loading-related cortical bone gain only in females. In addition to its role as a potential replacement for ERα, and modifier of ERα activity, the less well-studied ERß appears to facilitate rapid early effects of strain including activation of extracellular signal-regulated kinase and downregulation of Sost in well-differentiated cells of the osteoblast lineage including osteocytes. If these different roles are substantiated by further studies, it would appear that under normal circumstances ERα contributes primarily to the size and extent of bones' osteogenic response to load bearing through facilitating anabolic influences in osteoblasts and osteoblast progenitors, whereas ERß is more involved in the strain-related responses generated within resident cells including osteocytes.