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.

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.

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.

Loading-related regulation of transcription factor EGR2/Krox-20 in bone cells is ERK1/2 protein-mediated and prostaglandin, Wnt signaling pathway-, and insulin-like growth factor-I axis-dependent.

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression. In UMR106 cells, inhibition of ß1/ß3 integrin function had no effect on strain-related Egr2 expression, but it was inhibited by a COX2-selective antagonist and imitated by exogenous prostaglandin E2 (PGE2). This response to PGE(2) was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signaling, or LiCl had any effect on Egr2 mRNA expression, but it was increased by both insulin-like growth factor-1 and high, but not low, dose parathyroid hormone and exogenous Wnt-3a. The increases by strain, PGE2, Wnt-3a, and phorbol 12-myristate 13-acetate were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signaling pathways that constitute early responses of bone cells to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent "instructions" for adaptive (re)modeling is likely to depend upon their contextual activation, suppression, and interaction probably on more than one occasion.

Mechano-transduction in osteoblastic cells involves strain-regulated estrogen receptor alpha-mediated control of insulin-like growth factor (IGF) I receptor sensitivity to Ambient IGF, leading to phosphatidylinositol 3-kinase/AKT-dependent Wnt/LRP5 receptor-independent activation of beta-catenin signaling.

The capacity of bones to adjust their mass and architecture to withstand the loads of everyday activity derives from the ability of their resident cells to respond appropriately to the strains engendered. To elucidate the mechanisms of strain responsiveness in bone cells, we investigated in vitro the responses of primary mouse osteoblasts and UMR-106 osteoblast-like cells to a single period of dynamic strain. This stimulates a cascade of events, including activation of insulin-like growth factor I receptor (IGF-IR), phosphatidylinositol 3-kinase-mediated phosphorylation of AKT, inhibition of GSK-3beta, increased activation of beta-catenin, and associated lymphoid-enhancing factor/T cell factor-mediated transcription. Initiation of this pathway does not involve the Wnt/LRP5/Frizzled receptor and does not culminate in increased IGF transcription. The effect of strain on IGF-IR is mimicked by exogenous des-(1-3)IGF-I and is blocked by the IGF-IR inhibitor H1356. Inhibition of strain-related prostanoid and nitric oxide production inhibits strain-related (and basal) AKT activity, but their separate ectopic administration does not mimic it. Strain-related IGF-IR activation of AKT requires estrogen receptor alpha (ERalpha) with which IGF-1R physically associates. The ER blocker ICI 182,780 increases the concentration of des-(1-3)IGF-I necessary to activate this cascade, whereas estrogen inhibits both basal AKT activity and its activation by des-(1-3)IGF-I. These data suggest an initial cascade of strain-related events in osteoblasts in which strain activates IGF-IR, in association with ERalpha, so initiating phosphatidylinositol 3-kinase/AKT-dependent activation of beta-catenin and altered lymphoid-enhancing factor/T cell factor transcription. This cascade requires prostanoid/nitric oxide production and is independent of Wnt/LRP5.

Role of endocrine and paracrine factors in the adaptation of bone to mechanical loading.

There appears to be no unique mechanically sensitive pathway by which changes in bone loading regulate bone mass and architecture to ensure adequate structural strength. Rather, strain-derived changes in bone cells activate a number of nonspecific strain-sensitive pathways (including calcium fluxes, prostanoids, nitric oxide, extracellular signal-regulated kinase, and sclerostin), the activities of which are modified by a number of factors (including estrogen receptors) for which this contribution is subsidiary to other purposes. The strain-sensitive pathways modified by these factors interact with a number of other pathways, some of which appear to have specific osteoregulatory potential (eg, the parathyroid hormone pathway), whereas others such as the Wnt pathway appear to be associated primarily with the response mechanisms of proliferation, differentiation, and apoptosis. The outcome of these multiple interactions are stimuli for local bone formation, resorption, or maintenance of the status quo, to maintain existing bone architecture or adapt it to a new mechanical regimen.

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.

Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice.

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 microm. Treatment with iPTH (1-34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1-34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1-34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1-34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1-34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1-34) or either level of loading alone, whereas the combination of iPTH (1-34) and the "sufficient" level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1-34) in trabecular and cortical bone.

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.

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.

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.

Sympathetic nervous system does not mediate the load-induced cortical new bone formation.

UNLABELLED: The contribution of the SNS to bone's response to mechanical loading is unclear. Using a noninvasive model of axial loading of the murine tibia, we found that sciatic neurectomy enhances load-induced new cortical bone formation and that pharmacological blockade of the SNS does not affect such responses, indicating that the SNS does not mediate the osteogenic effects of loading in cortical bone. INTRODUCTION: There is increasing evidence that the sympathetic nervous system (SNS) contributes to the regulation of bone mass and may influence remodeling by modulating bones' response to mechanical load-bearing. The aim of this study was to examine the effect of sciatic neurectomy (SN) on the changes in cortical bone formation induced in response to mechanical loading and to investigate whether the SNS is directly involved in such load-induced responses. MATERIALS AND METHODS: Accordingly, load-induced responses were compared in tibias of growing and adult control C57Bl/J6 mice and in mice submitted to unilateral SN; noninvasive axial loading that induced 2,000 microstrain on the tibia lateral midshaft cortex was applied cyclically, 5 or 100 days after surgery, for 7 minutes, 3 days/week for 2 weeks, and mice received calcein on the third and last days of loading. Tibias were processed for histomorphometry, and transverse confocal images from diaphyseal sites were analyzed to quantify new cortical bone formation. Chemical SNS inactivation was achieved by prolonged daily treatment with guanethidine sulfate (GS) or by the introduction of propranolol in drinking water. RESULTS: Our results show that new cortical bone formation is enhanced by loading in all tibial sites examined and that load-induced periosteal and endosteal new bone formation was greater in the SN groups compared with sham-operated controls. This SN-related enhancement in load-induced cortical bone formation in tibias was more pronounced 100 days after neurectomy than after 5 days, suggesting that longer periods of immobilization promote a greater sensitivity to loading. In contrast, the increases in new bone formation induced in response to mechanical loading were similar in mice treated with either GS or propranolol compared with controls, indicating that inactivation of the SNS has no effect on load-induced cortical new bone formation. CONCLUSIONS: This study shows that SN, or the absence of loading function it entails, enhances loading-related new cortical bone formation in the tibia independently of the SNS.

Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element.

Systematic study of bones' responses to loading requires simple non-invasive models in appropriate experimental animals where the applied load is controllable and the changes in bone quantifiable. Herein, we validate a model for applying axial loads, non-invasively to murine tibiae. This allows the effects of mechanical loading in both cancellous and cortical bone to be determined within a single bone in which genetic, neuronal and functional influences can also be readily manipulated. Using female C57Bl/J6 mice, peak strains at the tibial mid-shaft were measured during walking (<300 micro epsilon tension) and jumping (<600 micro epsilon compression) with single longitudinally oriented strain gauges attached to the bone's lateral and medial surfaces. Identically positioned gauges were also used to determine, for calibration, the strains engendered by external applied compressive tibial loading between the flexed knee and ankle ex vivo. Applied loads between 5 and 13 N produced strains of 1150-2000 micro epsilon on the lateral surface, and in vivo repetitions of these loads on alternate days for 2 weeks produced significant load magnitude-related increases in cortical bone formation that were similar in mice at 8, 12 and 20 weeks of age. Micro-CT scans showed that loading significantly increases trabecular bone volume in 8 week old mice, but modifies trabecular organization with decreases in trabecular bone volume in 12 and 20 week old mice. This model for loading the tibia has several advantages over other approaches, including scope to study the effects of loading in cancellous as well as cortical bone, against a background of either disuse or of treatment with osteotropic agents within a single bone in normal, mutant and transgenic mice.

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.

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.

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.

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

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