Enhanced osteoclastic resorption and responsiveness to mechanical load in gap junction deficient bone

Emerging evidence suggests that connexin mediated gap junctional intercellular communication contributes to many aspects of bone biology including bone development, maintenance of bone homeostasis and responsiveness of bone cells to diverse extracellular signals. Deletion of connexin 43, the predominant gap junction protein in bone, is embryonic lethal making it challenging to examine the role of connexin 43 in bone in vivo. However, transgenic murine models in which only osteocytes and osteoblasts are deficient in connexin 43, and which are fully viable, have recently been developed. Unfortunately, the bone phenotype of different connexin 43 deficient models has been variable. To address this issue, we used an osteocalcin driven Cre-lox system to create osteoblast and osteocyte specific connexin 43 deficient mice. These mice displayed bone loss as a result of increased bone resorption and osteoclastogenesis. The mechanism underlying this increased osteoclastogenesis included increases in the osteocytic, but not osteoblastic, RANKL/OPG ratio. Previous in vitro studies suggest that connexin 43 deficient bone cells are less responsive to biomechanical signals. Interestingly, and in contrast to in vitro studies, we found that connexin 43 deficient mice displayed an enhanced anabolic response to mechanical load. Our results suggest that transient inhibition of connexin 43 expression and gap junctional intercellular communication may prove a potentially powerful means of enhancing the anabolic response of bone to mechanical loading.     

Mechanical Loading Synergistically Increases Trabecular Bone Volume and Improves Mechanical Properties in the Mouse when BMP Signaling Is Specifically Ablated in Osteoblasts

Bone homeostasis is affected by several factors, particularly mechanical loading and growth factor signaling pathways. There is overwhelming evidence to validate the importance of these signaling pathways, however, whether these signals work synergistically or independently to contribute to proper bone maintenance is poorly understood. Weight-bearing exercise increases mechanical load on the skeletal system and can improves bone quality. We previously reported that conditional knockout (cKO) of Bmpr1a, which encodes one of the type 1 receptors for Bone Morphogenetic Proteins (BMPs), in an osteoblast-specific manner increased trabecular bone mass by suppressing osteoclastogenesis. The cKO bones also showed increased cortical porosity, which is expected to impair bone mechanical properties. Here, we evaluated the impact of weight-bearing exercise on the cKO bone phenotype to understand interactions between mechanical loading and BMP signaling through BMPR1A. Male mice with disruption of Bmpr1a induced at 9 weeks of age, exercised 5 days per week on a motor-driven treadmill from 11 to 16 weeks of age. Trabecular bone volume in cKO tibia was further increased by exercise, whereas exercise did not affect the trabecular bone in the control genotype group. This finding was supported by decreased levels of osteoclasts in the cKO tibiae. The cortical porosity in the cKO bones showed a marginally significant decrease with exercise and approached normal levels. Exercise increased ductility and toughness in the cKO bones. Taken together, reduction in BMPR1A signaling may sensitize osteoblasts for mechanical loading to improve bone mechanical properties.     

Decreased pericellular matrix production and selection for enhanced cell membrane repair may impair osteocyte responses to mechanical loading in the aging skeleton

Transient plasma membrane disruptions (PMD) occur in osteocytes with in vitro and in vivo loading, initiating mechanotransduction. The goal here was to determine whether osteocyte PMD formation or repair is affected by aging. Osteocytes from old (24 months) mice developed fewer PMD (?76% females, ?54% males) from fluid shear than young (3 months) mice, and old mice developed fewer osteocyte PMD (?51%) during treadmill running. This was due at least in part to decreased pericellular matrix production, as studies revealed that pericellular matrix is integral to formation of osteocyte PMD, and aged osteocytes produced less pericellular matrix (?55%). Surprisingly, osteocyte PMD repair rate was faster (+25% females, +26% males) in osteocytes from old mice, and calcium wave propagation to adjacent nonwounded osteocytes was blunted, consistent with impaired mechanotransduction downstream of PMD in osteocytes with fast PMD repair in previous studies. Inducing PMD via fluid flow in young osteocytes in the presence of oxidative stress decreased postwounding cell survival and promoted accelerated PMD repair in surviving cells, suggesting selective loss of slower‐repairing osteocytes. Therefore, as oxidative stress increases during aging, slower‐repairing osteocytes may be unable to successfully repair PMD, leading to slower‐repairing osteocyte death in favor of faster‐repairing osteocyte survival. Since PMD are an important initiator of mechanotransduction, age‐related decreases in pericellular matrix and loss of slower‐repairing osteocytes may impair the ability of bone to properly respond to mechanical loading with bone formation. These data suggest that PMD formation and repair mechanisms represent new targets for improving bone mechanosensitivity with aging.     

Functions of the osteocyte network in the regulation of bone mass

Osteocytes establish an extensive intracellular and extracellular communication system via gap-junction-coupled cell processes and canaliculi throughout bone and the communication system is extended to osteoblasts on the bone surface. The osteocyte network is an ideal mechanosensory system and suitable for mechanotransduction. However, the overall function of the osteocyte network remains to be clarified, since bone resorption is enhanced by osteocyte apoptosis, which is followed by a process of secondary necrosis attributable to the lack of scavengers. The enhanced bone resorption is caused by the release of intracellular content, including immunostimulatory molecules that activate osteoclastogenesis through the canaliculi. Therefore, a mouse model is required in which the osteocyte network is disrupted but in which no bone resorption is induced, in order to evaluate the overall functions of the osteocyte network. One such model is the BCL2 transgenic mouse, in which the osteocyte network, including both intracellular and extracellular networks, is disrupted. Another model is the osteocyte-specific Gja1 knockout mouse, in which intercellular communication through gap junctions is impaired but the canalicular system is intact. Combining the findings from these mouse models with previous histological observations showing the inverse linkage between osteocyte density and bone formation, we conclude that the osteocyte network enhances bone resorption and inhibits bone formation under physiological conditions. Further, studies with BCL2 transgenic mice show that these osteocyte functions are augmented in the unloaded condition. In this condition, Rankl upregulation in osteoblasts and Sost upregulation in osteocytes are, at least in part, responsible for enhanced bone resorption and suppressed bone formation, respectively.     

Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone‐specific Pdpn knockdown on osteocyte form and function in the post‐natal mouse. Extensive skeletal phenotyping of male and female 6‐week‐old Oc‐cre;Pdpn^flox/flox (cKO) mice and their Pdpn^flox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age‐matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.     

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.     

Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.     

Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice

X-linked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wild-type (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counterregulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice.     

Conditional disruption of IGF-I gene in type 1α collagen-expressing cells shows an essential role of IGF-I in skeletal anabolic response to loading

To establish a causal role for locally produced IGF-I in the mechanical strain response in the bone, we have generated mice with conditional disruption of the insulin-like growth factor (IGF) I gene in type 1α(2) collagen-expressing cells using the Cre-loxP approach. At 10 wk of age, loads adjusted to account for bone size difference were applied via four-point bending or axial loading (AL) in mice. Two wk of bending and AL produced significant increases in bone mineral density and bone size at the middiaphysis of wild-type (WT), but not knockout (KO), mice. In addition, AL produced an 8-25% increase in trabecular parameters (bone volume-tissue volume ratio, trabecular thickness, and trabecular bone mineral density) at the secondary spongiosa of WT, but not KO, mice. Histomorphometric analysis at the trabecular site revealed that AL increased osteoid width by 60% and decreased tartrate-resistance acidic phosphatase-labeled surface by 50% in the WT, but not KO, mice. Consistent with the in vivo data, blockade of IGF-I action with inhibitory IGF-binding protein (IGFBP4) in vitro completely abolished the fluid flow stress-induced MC3T3-E1 cell proliferation. One-way ANOVA revealed that expression levels of EFNB1, EFNB2, EFNA2, EphB2, and NR4a3 were different in the loaded bones of WT vs. KO mice and may, in part, be responsible for the increase in bone response to loading in the WT mice. In conclusion, IGF-I expressed in type 1 collagen-producing bone cells is critical for converting mechanical signal to anabolic signal in bone, and other growth factors cannot compensate for the loss of local IGF-I.     

Deletion of Mbtps1 (Pcsk8, S1p,Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10–12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.     

Thermally Induced Osteocyte Damage Initiates a Remodelling Signaling Cascade

Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47°C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47°C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis followed by a cascade of remodelling responses.     

Inhibition of Osteocyte Apoptosis Prevents the Increase in Osteocytic Receptor Activator of Nuclear Factor κB Ligand (RANKL) but Does Not Stop Bone Resorption or the Loss of Bone Induced by Unloading

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.     

Death of osteocytes turns off the inhibition of osteoclasts and triggers local bone resorption

Osteocytes have been suggested to play a role in the regulation of bone resorption, although their effect on bone turnover has remained controversial. In order to study this open question, we developed an organ culture system based on isolated rat calvaria, where the osteocyte viability and its effect on osteoclastic bone resorption can be monitored. Our results suggest that osteocytes are constitutively negative regulators of osteoclastic activity. Osteoclasts, which were cultured on calvarial slices with living osteocytes inside, failed to form actin rings which are the hallmarks of resorbing cells. A similar inhibitory effect was also achieved by the conditioned medium obtained from calvarial organ culture, suggesting that living osteocytes produce yet unrecognized osteoclast inhibitors. On the contrary, when osteocyte apoptosis was induced, this inhibitory effect disappeared and strong osteoclastic bone resorption activity was observed. Thus, local apoptosis of osteocytes may play a major role in triggering local bone remodeling.     

Enhanced periosteal and endocortical responses to axial tibial compression loading in conditional connexin43 deficient mice

The gap junction protein, connexin43 (Cx43) is involved in mechanotransduction in bone. Recent studies using in vivo models of conditional Cx43 gene (Gja1) deletion in the osteogenic linage have generated inconsistent results, with Gja1 ablation resulting in either attenuated or enhanced response to mechanical load, depending upon the skeletal site examined or the type of load applied. To gain further insights on Cx43 and mechanotransduction, we examined bone formation response at both endocortical and periosteal surfaces in 2-month-old mice with conditional Gja1 ablation driven by the Dermo1 promoter (cKO). Relative to wild type (WT) littermates, it requires a larger amount of compressive force to generate the same periosteal strain in cKO mice. Importantly, cKO mice activate periosteal bone formation at a lower strain level than do WT mice, suggesting an increased sensitivity to mechanical load in Cx43 deficiency. Consistently, trabecular bone mass also increases in mutant mice upon load, while it decreases in WT. On the other hand, bone formation actually decreases on the endocortical surface in WT mice upon application of axial mechanical load, and this response is also accentuated in cKO mice. These changes are associated with increase of Cox-2 in both genotypes and further decrease of Sost mRNA in cKO relative to WT bones. Thus, the response of bone forming cells to mechanical load differs between trabecular and cortical components, and remarkably between endocortical and periosteal envelopes. Cx43 deficiency enhances both the periosteal and endocortical response to mechanical load applied as axial compression in growing mice.     

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.