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Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the pathway's activity in the skeleton, cause high bone mass in human patients and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive bossing of the forehead and mandibular overgrowth occur in almost all patients. Treatments that would provide symptomatic relief in these patients are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the Frizzled receptor. Chemical inhibition of porcupine is a highly selective inhibitor of all Wnt signaling. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling: homozygosity for loss-of-function in SOST, which models Sclerosteosis, and two strains of mice carrying different point mutations in LRP5 (equivalent to human G171V and A214V) with porcupine inhibitors for 5–6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in patients who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.

Loss-of-function mutations in the Sost gene lead to high bone mass phenotypes. Pharmacological inhibition of Sost/sclerostin provides a new drug strategy for treating osteoporosis. Questions remain as to how physical activity may affect bone mass under sclerostin inhibition and if that effect differs between males and females. We previously observed in female Sost knockout (KO) mice an enhanced cortical bone formation response to a moderate level of applied loading (900 με at the tibial midshaft). The purpose of the present study was to examine cortical bone adaptation to the same strain level applied to male Sost KO mice. Strain-matched in vivo compressive loading was applied to the tibiae of 10-, 26- and 52-week-old male Sost KO and littermate control (LC) mice. The effect of tibial loading on bone (re)modeling was measured by microCT, 3D time-lapse in vivo morphometry, 2D histomorphometry and gene expression analyses. As expected, Sost deficiency led to high cortical bone mass in 10- and 26-week-old male mice as a result of increased bone formation. However, the enhanced bone formation associated with Sost deficiency did not appear to diminish with skeletal maturation. An increase in bone resorption was observed with skeletal maturation in male LC and Sost KO mice. Two weeks of in vivo loading (900 με at the tibial midshaft) induced only a mild anabolic response in 10- and 26-week-old male mice, independent of Sost deficiency. A decrease in the Wnt inhibitor Dkk1 expression was observed 3 h after loading in 52-week-old Sost KO and LC mice, and an increase in Lef1 expression was observed 8 h after loading in 10-week-old Sost KO mice. The current results suggest that long-term inhibition of sclerostin in male mice does not influence the adaptive response of cortical bone to moderate levels of loading. In contrast with our previous strain-matched study in females showing enhanced bone responses with Sost ablation, these results in males indicate that the influence of Sost deficiency on the cortical bone formation response to a moderate level of loading differs between males and females. Clinical studies examining antibodies to inhibit sclerostin may need to consider that the efficacy of additional physical activity regimens may be sex dependent.

A new therapeutic option to treat osteoporosis is focused on Wnt signaling and its inhibitor sclerostin, a product of the Sost gene. In this work, we study the effect of sclerostin deficiency on trabecular bone formation and resorption in male and female mice and whether it affects mechano-responsiveness. Male and female 10- and 26-week-old Sost knockout (KO) and littermate controls (LCs) were subjected to in vivo mechanical loading of the left tibia for 2 weeks. The right tibia served as internal control. The mice were imaged using in vivo micro-computed tomography at days 0, 5, 10, and 15 and tibiae were collected for histomorphometric analyses after euthanasia. Histomorphometry and micro-CT-based 3D time-lapse morphometry revealed an anabolic and anti-catabolic effect of Sost deficiency although increased trabecular bone resorption accompanied by diminished trabecular bone formation occurred with age. Loading led to diminished resorption in adult female but not in male mice. A net gain in bone volume could be achieved with mechanical loading in Sost KO adult female mice, which occurred through a further reduction in resorbed bone volume. Our data show that sclerostin deficiency has a particularly positive effect in adult female mice. Sclerostin antibodies are approved to treat postmenopausal women with high risk of osteoporotic fractures. Further studies are required to clarify whether both sexes benefit equally from sclerostin inhibition.

Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043—a derivative of angiopoietin-like 3 (ANGPTL3)—as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5β1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial (NCT02491281; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial (NCT04864392) in patients with knee OA.

Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth’s mesosphere.

Decreased activity or expression of sclerostin, an endogenous inhibitor of Wnt/β-catenin signaling, results in increased bone formation and mass. Antibodies targeting and neutralizing sclerostin (Scl-Ab) have been shown to increase bone mass and reduce fracture risk. Sclerostin is also important in modulating the response of bone to changes in its biomechanical environment. However, the effects of Scl-Ab on mechanotransduction are unclear, and it was speculated that the loading response may be altered for individuals receiving Scl-Ab therapy. To address this, we carried out a 2 week study of tibial cyclic compressive loading on C57Bl/6 mice treated with vehicle or 100mg/kg/week Scl-Ab. Increases in bone volume, density, and dynamic bone formation were seen with loading, and the anabolic response was further increased by the combination of load and Scl-Ab. To investigate the underlying mechanism, gene profiling by RNA sequencing (RNAseq) was performed on tibiae isolated from mice from all four experimental groups. Major alterations in Wnt/β-catenin gene expression were seen with tibial loading, however not with Scl-Ab treatment alone. Notably, the combination of load and Scl-Ab elicited a synergistic response from a number of specific Wnt-related and mechanotransduction factors. An unexpected finding was significant upregulation of factors in the Rho GTPase signaling pathway with combination treatment. In summary, combination therapy had a more profound anabolic response than either Scl-Ab or loading treatment alone. The Wnt/β-catenin and Rho GTPase pathways were implicated within bone mechanotransduction, and support the concept that bone mechanotransduction is likely to encompass a number of interconnected signaling pathways. This article is protected by copyright. All rights reserved

In this study, we examined the therapeutic potential of anti-Sclerostin Antibody (Scl-Ab) and bisphosphonate treatments for the bone fragility disorder Osteogenesis Imperfecta (OI). Mice with the Amish OI mutation (Col1a2 G610C mice) and control wild type littermates (WT) were treated from week 5 to week 9 of life with (1) saline (control), (2) zoledronic acid given 0.025mg/kg s.c. weekly (ZA), (3) Scl-Ab given 50mg/kg IV weekly (Scl-Ab), or (4) a combination of both (Scl-Ab/ZA). Functional outcomes were prioritized and included bone mineral density (BMD), bone microarchitecture, long bone bending strength, and vertebral compression strength. By dual-energy absorptiometry, Scl-Ab treatment alone had no effect on tibial BMD, while ZA and Scl-Ab/ZA significantly enhanced BMD by week 4 (+16% and +27% respectively, P0.05). Scl-Ab/ZA treatment also led to increases in cortical thickness and tissue mineral density, and restored the tibial 4-point bending strength to that of control WT mice. In the spine, all treatments increased compression strength over controls, but only the combined group reached the strength of WT controls. Scl-Ab showed greater anabolic effects in the trabecular bone than in cortical bone. In summary, the Scl-Ab/ZA intervention was superior to either treatment alone in this OI mouse model, however further studies are required to establish its efficacy in other preclinical and clinical scenarios.

Career situation of first and presenting author Student for a master or a PhD. Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Sclerostin loss-of-function leads to a higher bone mass and bone strength. Therefore, inhibition of sclerostin is currently considered as a promising treatment for osteoporosis. Surprisingly, in a TNFα-dependent arthritis mouse model (hTNFtg) the genetic deficiency of sclerostin caused a deterioration of disease severity. hTNFtg mice lacking sclerostin displayed enhanced and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives In order to understand the underlying mechanisms, we aimed to investigate the direct and indirect impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost-/-) mice were crossbred with hTNFtg mice to obtain sost-/-/hTNFtg mice, from which synovial fibroblasts (SF) were isolated. Cocultures of synovial fibroblasts and green fluorescent protein (GFP+) bone marrow-derived macrophages (BMM) were performed and osteoclastogenesis was analysed. Receptor activator of NF-kB ligand (RANKL) and macrophage colony stimulating factor (MCSF) expression was measured by ELISA and IL-1a expression by Western Blot. Viability of osteoclasts precursors was measured by MTT Assay. Results In cocultures of SF and GFP+ BMM, osteoclast formation was enhanced by sost-/-/hTNFtg SF compared to hTNFtg SF. Expression of RANKL and MCSF, two crucial factors for osteoclast differentiation, was not different between the genotypes. Interestingly, stimulation of wildtype BMM with conditioned media (CM) from hTNFtg or sost-/-/hTNFtg synovial fibroblasts showed no TRAP+ cells at all. However, CM supplemented with RANKL lead to an elevated number of osteoclasts using sost-/-/hTNFtg CM compared to hTNFtg CM, pointing to a secreted factor promoting osteoclast development. In this regard, the osteogenic factor IL-1α, was higher expressed in sost-/-/hTNFtg SF than in hTNFtg SF. Moreover, the treatment of cocultures with recombinant sclerostin lead to a decreased number of osteoclasts in both genotypes. Accordingly, sclerostin inhibited osteoclastogenesis in monocultures when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin mainly on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg SF promotes RANKL-mediated osteoclast differentiation, which is likely dependent on the inhibitory effect of sclerostin itself and/or on the promoting effect of higher levels of IL-1α. Disclosure of Interest None declared.

Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/β-catenin pathway can overcome radiation-induced DNA damage and apoptosis of osteoblastic cells. Neutralization of circulating sclerostin with a monoclonal antibody (Scl-Ab) is an innovative approach for treating osteoporosis by enhancing Wnt/β-catenin signaling in bone. Together with the fact that focal radiation increases sclerostin amount in bone, we sought to determine whether weekly treatment with Scl-Ab would prevent focal radiotherapy-induced osteoporosis in mice. Micro-CT and histomorphometric analyses demonstrated that Scl-Ab blocked trabecular bone structural deterioration after radiation by partially preserving osteoblast number and activity. Consistently, trabecular bone in sclerostin null mice was resistant to radiation via the same mechanism. Scl-Ab accelerated DNA repair in osteoblasts after radiation by reducing the number of γ-H2AX foci, a DNA double-strand break marker, and increasing the amount of Ku70, a DNA repair protein, thus protecting osteoblasts from radiation-induced apoptosis. In osteocytes, apart from using similar DNA repair mechanism to rescue osteocyte apoptosis, Scl-Ab restored the osteocyte canaliculi structure that was otherwise damaged by radiation. Using a lineage tracing approach that labels all mesenchymal lineage cells in the endosteal bone marrow, we demonstrated that radiation damage to mesenchymal progenitors mainly involves shifting their fate to adipocytes and arresting their proliferation ability but not inducing apoptosis, which are different mechanisms from radiation damage to mature bone forming cells. Scl-Ab treatment partially blocked the lineage shift but had no effect on the loss of proliferation potential. Taken together, our studies provide proof-of-principle evidence for a novel use of Scl-Ab as a therapeutic treatment for radiation-induced osteoporosis and establish molecular and cellular mechanisms that support such treatment. © 2016 American Society for Bone and Mineral Research.

Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice.

Sclerostin, a product of the Sost gene, is a Wnt-inhibitor and thus negatively regulates bone accrual. Canonical Wnt/β-catenin signalling is also known to be activated in mechanotransduction. Sclerostin neutralizing antibodies are being tested in ongoing clinical trials to target osteoporosis and osteogenesis imperfecta but their interaction with mechanical stimuli on bone formation remains unclear. Sost knockout (KO) mice were examined to gain insight into how long-term Sost deficiency alters the local mechanical environment within the bone. This knowledge is crucial as the strain environment regulates bone adaptation. We characterized the bone geometry at the tibial midshaft of young and adult Sost KO and age-matched littermate control (LC) mice using microcomputed tomography imaging. The cortical area and the minimal and maximal moment of inertia were higher in Sost KO than in LC mice, whereas no difference was detected in either the anterior–posterior or medio-lateral bone curvature. Differences observed between age-matched genotypes were greater in adult mice. We analysed the local mechanical environment in the bone using finite-element models (FEMs), which showed that strains in the tibiae of Sost KO mice are lower than in age-matched LC mice at the diaphyseal midshaft, a region commonly used to assess cortical bone formation and resorption. Our FEMs also suggested that tissue mineral density is only a minor contributor to the strain distribution in tibial cortical bone from Sost KO mice compared to bone geometry. Furthermore, they indicated that although strain gauging experiments matched strains at the gauge site, strains along the tibial length were not comparable between age-matched Sost KO and LC mice or between young and adult animals within the same genotype.

Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. The lack of sclerostin in humans and mice leads to a higher bone mass and bone strength known as sclerosteosis. Therefore, inhibition of sclerostin is currently investigated as a treatment against osteoporosis. Surprisingly, the genetic deficiency or pharmacological inhibition of sclerostin causes a deterioration of disease severity in a TNFα-dependent arthritis mouse model (hTNFtg). hTNFtg mice lacking sclerostin displayed enhanced joint inflammation, cartilage loss and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives We want to investigate the impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost -/- ) mice were crossbred with hTNFtg mice to obtain sost -/- /hTNFtg synovial fibroblasts. Co-cultures of synovial fibroblasts and wildtype bone marrow macrophages were analysed by TRAP staining. RANKL expression was measured by ELISA and cytokine expression by array analysis and Western Blot. Moreover, the influence of sclerostin on osteoclastogenesis was additionally analysed in mono-cultures. Results In our co-culture system of synovial fibroblasts and bone marrow derived macrophages, fibroblasts from sost -/- /hTNFtg mice strongly promote osteoclastogenesis in comparison to hTNFtg synovial fibroblasts. Notably, no increased expression of receptor activator of NF-kB ligand (RANKL) was detectable in sost -/- /hTNFtg fibroblasts even after stimulation with inflammatory cytokines. Interestingly, basal secretion of IL-1α, which is known to stimulate osteoclastogenesis, was higher in sost -/- /hTNFtg compared to hTNFtg fibroblasts. Accordingly, sclerostin inhibited osteoclastogenesis when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg synovial fibroblasts promotes RANKL-mediated osteoclastogenesis, which is most likely dependent on IL-1α. Disclosure of interest None declared

Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/β-catenin pathway and decreased expression of either results in increased bone formation and mass. As both affect the same signaling pathway, we aimed to elucidate the redundancy and/or compensation of sclerostin and DKK1. Weekly sclerostin antibody (Scl-Ab) was used to treat 9-week-old female Dkk1 KO (Dkk1-/-:Wnt3+/-) mice and compared to Scl-Ab-treated wild-type mice as well as vehicle-treated Dkk1 KO and wild-type animals. While Wnt3 heterozygote (Wnt3+/-) mice show no bone phenotype, Scl-Ab and vehicle-treated control groups of this genotype were included. Specimens were harvested after 3 weeks for microCT, bone histomorphometry, anti-sclerostin immunohistochemistry, and biomechanical testing. Scl-Ab enhanced bone anabolism in all treatment groups, but with synergistic enhancement seen in the cancellous compartment of Dkk1 KO mice (bone volume + 55% Dkk1 KO p

Hydrogels are under active development for controlled drug delivery, but their clinical translation has been limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg/mL) over four weeks. The release rate was primarily governed by ionic exchange and could be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury was used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine. [46,47,48]

Chronic Kidney Disease (CKD) is associated with abnormalities in bone quantity and quality leading to increased fractures. Recent studies suggest abnormalities of Wnt signaling in animal models of CKD and elevated sclerostin levels in patients with CKD. The goal of this study was to evaluate the effectiveness of anti-sclerostin antibody treatment in an animal model of progressive CKD with low and high parathyroid hormone (PTH) levels. Cy/+ male rats (CKD) were treated without or with calcium in the drinking water at 25 weeks of age to stratify the animals into high PTH and low PTH groups, respectively, by 30 weeks. Animals were then treated with anti-sclerostin antibody at 100 mg/kg IV weekly for 5 doses, a single 20 ug/kg subcutaneous dose of zoledronic acid, or no treatment and sacrificed at 35 weeks. As a positive control, the efficacy of anti-sclerostin antibody treatment was also evaluated in normal littermates. The results demonstrated that the CKD animals with high PTH had lower calcium, higher phosphorus, and lower FGF23 compared to the CKD animals with low PTH. Treatment with anti-sclerostin Ab had no effect on any of the biochemistries, while zoledronic acid lowered dkk-1 levels. The anti-sclerostin antibody increased trabecular BV/TV., trabecular mineralization surface, in animals with low, but not high, PTH. Neither anti-sclerostin antibody nor zoledronic acid improved biomechanical properties in the animals. Cortical porosity was severe in high PTH animals and unaffected by either treatment. In contrast, in normal animals treated with anti-sclerostin antibody, there was an improvement in bone volume, cortical geometry, and biomechanical properties. In summary, this is the first study to test the efficacy of anti-sclerostin Ab treatment on animals with advanced CKD. We found efficacy in improving bone properties only when the PTH levels were low.

Decreased activity or expression of sclerostin, an endogenous inhibitor of Wnt/β-catenin signaling, results in increased bone formation and mass. Antibodies targeting and neutralizing sclerostin (Scl-Ab) have been shown to increase bone mass and reduce fracture risk. Sclerostin is also important in modulating the response of bone to changes in its biomechanical environment. However, the effects of Scl-Ab on mechanotransduction are unclear, and it was speculated that the loading response may be altered for individuals receiving Scl-Ab therapy. To address this, we carried out a 2-week study of tibial cyclic compressive loading on C57Bl/6 mice treated with vehicle or 100 mg/kg/wk Scl-Ab. Increases in bone volume, density, and dynamic bone formation were found with loading, and the anabolic response was further increased by the combination of load and Scl-Ab. To investigate the underlying mechanism, gene profiling by RNA sequencing (RNAseq) was performed on tibias isolated from mice from all four experimental groups. Major alterations in Wnt/β-catenin gene expression were found with tibial loading, however not with Scl-Ab treatment alone. Notably, the combination of load and Scl-Ab elicited a synergistic response from a number of specific Wnt-related and mechanotransduction factors. An unexpected finding was significant upregulation of factors in the Rho GTPase signaling pathway with combination treatment. In summary, combination therapy had a more profound anabolic response than either Scl-Ab or loading treatment alone. The Wnt/β-catenin and Rho GTPase pathways were implicated within bone mechanotransduction and support the concept that bone mechanotransduction is likely to encompass a number of interconnected signaling pathways. © 2017 American Society for Bone and Mineral Research.

Sclerostin is a soluble antagonist of canonical Wnt signaling and a strong inhibitor of bone formation. We present experimental data on the role of sclerostin in chronic kidney disease - bone mineral disorder (CKD-MBD). Methods We performed 5/6 nephrectomies in 36-week-old sclerostin-deficient (SOST−/−) B6-mice and in C57BL/6J wildtype (WT) mice. Animals received a high phosphate diet for 11 weeks. The bones were analyzed by high-resolution micro-computed tomography (μCT) and quantitative bone histomorphometry. Aortic tissue was analyzed regarding the extent of vascular calcification. Results All nephrectomized mice had severe renal failure, and parathyroid hormone was highly increased compared to corresponding sham animals. All SOST−/− animals revealed the expected high bone mass phenotype. Overall, the bone compartment in WT and SOST−/− mice responded similarly to nephrectomy. In uremic WT animals, μCT data at both the distal femur and lumbar spine revealed significantly increased trabecular volume compared to non-uremic WTs. In SOST−/− mice, the differences between trabecular bone volume were less pronounced when comparing uremic with sham animals. Cortical thickness and cortical bone density at the distal femur decreased significantly and comparably in both genotypes after 5/6 nephrectomy compared to sham animals (cortical bone density − 18% and cortical thickness − 32%). Overall, 5/6 nephrectomy and concomitant hyperparathyroidism led to a genotype-independent loss of cortical bone volume and density. Overt vascular calcification was not detectable in either of the genotypes. Conclusion Renal osteodystrophy changes were more pronounced in WT mice than in SOST−/− mice. The high bone mass phenotype of sclerostin deficiency was detectable also in the setting of chronic renal failure with severe secondary hyperparathyroidism. Abbreviations α-SMA, alpha smooth muscle actin; AjAR, adjusted apposition rate; B.Ar, bone area; BFR, bone formation rate; cbfa1, core binding factor 1; CKD-MBD, chronic kidney disease and mineral bone disorder; EPm, eroded perimeter; Ntx, nephrectomy; O.Ar, osteoid area; Ob, osteoblast perimeter; Oc, osteoclast perimeter; O.Wi, osteoid width; PTH, parathyroid hormone; s.c., subcutaneous; VSMCs, vascular smooth muscle cells; WT, wildype

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.

The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications.

This chapter is divided into three subsections: The bone biology part will provide some insight into the matrix composition as well as the origin and basic function attributed to the cellular components, the osteoblasts osteocytes and osteoclasts. In the second section, the interplay between these matrix components and the cells will be discussed in the context of skeletal growth, skeletal adaptation (modeling), and skeletal maintenance (bone remodeling). Finally, the third section will address similarities and differences in bone biology between human, nonhuman primate, rat, and mouse, the most commonly used species in the study of bone metabolic disorders. Other large animals like dogs, sheep, mini-pigs, or rabbits which are being successfully used to study orthopedic conditions (implant ingrowth, fracture healing, and bone augmentation) have not been addressed in this chapter.

Bone adaptation optimizes mass and structure, but the mechano-response is already reduced at maturation. Downregulation of sclerostin was believed to be a mandatory step in mechano-adaptation, but in young mice it was shown that load-induced formation can occur independent of sclerostin, a product of the Sost gene. We hypothesized that the bone formation and resorption response to loading is not affected by Sost deficiency, but is age-specific. Our findings indicate that the anabolic response to in vivo tibial loading was reduced at maturation in Sost Knockout (KO) and littermate control (LC) mice. Age affected all anabolic and catabolic parameters and altered Sost and Wnt target gene expression. While load-induced cortical resorption was similar between genotypes, loading-induced gains in mineralizing surface was enhanced in Sost KO compared to LC mice. Loading led to a downregulation in expression of the Wnt inhibitor Dkk1. Expression of Dkk1 was greater in both control and loaded limbs of Sost KO compared to LC mice suggesting a compensatory role in the absence of Sost. These data suggest physical activity could enhance bone mass concurrently with sclerostin-neutralizing antibodies, but treatment strategies should consider the influence of age on ultimate load-induced bone mass gains.

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long-term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost−/− and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (−9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost−/− tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost−/− mice over their normal load controls. The increased BV was associated with significantly increased mid-shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost−/− mice than in wild type controls. Thus, long-term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load. © 2014 American Society for Bone and Mineral Research.

High bone mass in animals and humans with sclerostin deficiency is associated with increased bone strength, which is not the case for all disorders with high bone mineral density, some of which are even associated with fragility fractures owing to unfavorable bone composition. In the current study we investigated whether alterations in bone composition may contribute to the bone strength characteristics associated with lack of sclerostin. We examined cortical bone of Sost-knockout (KO) mice (n = 9, 16 weeks old) and sclerosteosis patients (young [4 to 14 years], n = 4 and adults [24 and 43 years], n = 2) by quantitative backscattered electron imaging and Raman microspectroscopy and compared it to bone from wild-type mice and healthy subjects, respectively. In Sost-KO mice endocortical bone exhibited altered bone composition, whereas subperiosteal bone was unchanged. When comparing endocortical bone tissue of identical tissue age as defined by sequential dual fluorochrome labeling the average bone matrix mineralization was reduced -1.9% (p < 0.0001, younger tissue age) and -1.5% (p < 0.05, older tissue age), and the relative proteoglycan content was significantly increased. Similarly, bone matrix mineralization density distribution was also shifted toward lower matrix mineralization in surgical samples of compact bone of sclerosteosis patients. This was associated with an increase in mineralization heterogeneity in the young population. In addition, and consistently, the relative proteoglycan content was increased. In conclusion, we observed decreased matrix mineralization and increased relative proteoglycan content in bone subcompartments of Sost-KO mice-a finding that translated into sclerosteosis patients. We hypothesize that the altered bone composition contributes to the increased bone strength of patients with sclerostin deficiency.

Antagonism of the calcium-sensing receptor in the parathyroid gland leads to parathyroid hormone (PTH) release. Calcilytics are a new class of molecules designed to exploit this mechanism. In order to mimic the known bone-anabolic pharmacokinetic (PK) profile of s.c. administered PTH, such molecules must trigger sharp, transient and robust release of PTH. The results of two early clinical studies with the orally-active calcilytic AXT914, a quinazolin-2ne derivative are reported. These were GCP-compliant, single and multiple dose studies of PK/PD and tolerability in healthy volunteers and postmenopausal women. The first study, examined single ascending doses (4 to 120 mg) and limited multiple doses (60 or 120 mg q.d. for 12 days) of AXT914. The second study was a randomized, double-blind, active- and placebo-controlled, 4-week repeat-dose parallel group study of healthy postmenopausal women (45 and 60 mg AXT914, placebo, 20 μg Forteo/teriparatide/PTH(1–34) fragment). AXT914 was well tolerated at all doses and reproducibly induced the desired PTH-release profiles. Yet, 4 weeks of 45 or 60 mg AXT914 did not result in the expected changes in circulating bone biomarkers seen with teriparatide. However total serum calcium levels increased above baseline in the 45 and 60 mg AXT914 treatment groups (8.0 % and 10.7%, respectively), compared to that in the teriparatide and placebo groups (1.3% and 1.0%, respectively). Thus the trial was terminated after a planned interim analysis due to lack of effect on bone formation biomarkers and dose-limiting effects on serum calcium. In conclusion, AXT914 was well tolerated but the observed transient and reproducible PTH-release after repeat oral administration of AXT914 which showed an exposure profile close to that of s c. PTH, did not translate into a bone anabolic response and was associated with a persistent dose-related increase in serum calcium concentrations.

Sclerostin is a Wnt signalling antagonist that controls bone metabolism. Sclerostin is expressed by osteocytes and cementocytes; however, its role in the formation of dental structures remains unclear. Here, we analysed the mandibles of sclerostin knockout mice to determine the influence of sclerostin on dental structures and dimensions using histomorphometry and micro-computed tomography (μCT) imaging. μCT and histomorphometric analyses were performed on the first lower molar and its surrounding structures in mice lacking a functional sclerostin gene and in wild-type controls. μCT on six animals in each group revealed that the dimension of the basal bone as well as the coronal and apical part of alveolar part increased in the sclerostin knockout mice. No significant differences were observed for the tooth and pulp chamber volume. Descriptive histomorphometric analyses of four wild-type and three sclerostin knockout mice demonstrated an increased width of the cementum and a concomitant moderate decrease in the periodontal space width. Taken together, these results suggest that the lack of sclerostin mainly alters the bone and cementum phenotypes rather than producing abnormalities in tooth structures such as dentin.

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low-density lipoprotein receptor-related protein (LRP) 5/6 Wnt co-receptors, thereby inhibiting Wnt/β-catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis-pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost(-/-) ), Lrp5 (Lrp5(-/-) ), or both (Sost(-/-) ;Lrp5(-/-) ) to elucidate the mechanism of action of Sost in vivo. Sost deficiency-induced bone gain was significantly blunted in Sost(-/-) ;Lrp5(-/-) mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost(-/-) ;Lrp5(-/-) mice and most bone parameters were elevated relative to wild-type. To test whether the remaining bone increases in Sost(-/-) ;Lrp5(-/-) animals depend on Lrp6, we treated wild-type, Sost(-/-) , and Sost(-/-) ;Lrp5(-/-) mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class-mediated Lrp6 signaling reduced cancellous bone mass and density in wild-type mice. Surprisingly, it reversed the abnormal bone gain in Sost(-/-) and Sost(-/-) ;Lrp5(-/-) mice to wild-type levels irrespective of enhancement or blockage of Wnt3a class-mediated Lrp6 activity. Thus, whereas Sost deficiency-induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class-induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss-of-function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options.

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases.

Activating FGFR3 mutations in human result in achondroplasia (ACH), the most frequent form of dwarfism, where cartilages are severely disturbed causing long bones, cranial base and vertebrae defects. Because mandibular development and growth rely on cartilages that guide or directly participate to the ossification process, we investigated the impact of FGFR3 mutations on mandibular shape, size and position. By using CT scan imaging of ACH children and by analyzing Fgfr3Y367C/+ mice, a model of ACH, we show that FGFR3 gain-of-function mutations lead to structural anomalies of primary (Meckel's) and secondary (condylar) cartilages of the mandible, resulting in mandibular hypoplasia and dysmorphogenesis. These defects are likely related to a defective chondrocyte proliferation and differentiation and pan-FGFR tyrosine kinase inhibitor NVP-BGJ398 corrects Meckel's and condylar cartilages defects ex vivo. Moreover, we show that low dose of NVP-BGJ398 improves in vivo condyle growth and corrects dysmorphologies in Fgfr3Y367C/+ mice, suggesting that postnatal treatment with NVP-BGJ398 mice might offer a new therapeutic strategy to improve mandible anomalies in ACH and others FGFR3-related disorders.

Item does not contain fulltext Sclerostin, an inhibitor of the Wnt/beta-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFalpha (tumor necrosis factor alpha)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFalpha transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFalpha-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFalpha signaling. Sclerostin effectively blocked TNFalpha- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFalpha-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFalpha-dependent comorbidities.

Myocyte enhancer factors 2 (MEF2) are required for expression of the osteocyte bone formation inhibitor Sost in vitro, implying these transcription factors in bone biology. Here, we analyzed the in vivo function of Mef2c in osteocytes in male and female mice during skeletal growth and aging. Dmp1-Cre-induced Mef2c deficiency led to progressive decreases in Sost expression by 40% and 70% in femoral cortical bone at 3.5 months and 5 to 6 months of age. From 2 to 3 months onward, bone mass was increased in the appendicular and axial skeleton of Mef2c mutant relative to control mice. Cortical thickness and long bone and vertebral trabecular density were elevated. To assess whether the increased bone mass was related to the decreased Sost expression, we characterized 4-month-old heterozygous Sost-deficient mice. Sost heterozygotes displayed similar increases in long bone mass and density as Mef2c mutants, but the relative increases in axial skeletal parameters were mostly smaller. At the cellular level, bone formation parameters were normal in 3.5-month-old Mef2c mutant mice, whereas bone resorption parameters were significantly decreased. Correspondingly, cortical expression of the anti-osteoclastogenic factor and Wnt/β-catenin target gene osteoprotegerin (OPG) was increased by 70% in Mef2c mutant males. Furthermore, cortical expression of the Wnt signaling modulators Sfrp2 and Sfrp3 was strongly deregulated in both sexes. In contrast, heterozygous Sost deficient males displayed mildly increased osteoblastic mineral apposition rate, but osteoclast surface and cortical expression of osteoclastogenic regulators including OPG were normal and Sfrp2 and Sfrp3 were not significantly changed. Together, our data demonstrate that Mef2c regulates cortical Sfrp2 and Sfrp3 expression and is required to maintain normal Sost expression in vivo. Yet, the increased bone mass phenotype of Mef2c mutants is not directly related to the reduced Sost expression. We identified a novel function for Mef2c in control of adult bone mass by regulation of osteoclastic bone resorption.

Parathyroid hormone (PTH), when injected daily as either the intact hormone PTH(1-84) or the active fragment PTH(1-34) (teriparatide), is an efficacious bone anabolic treatment option for osteoporosis patients. Injections lead to rapid and transient spikes in hormone exposure levels, a profile which is a prerequisite to effectively form bone. Oral antagonists of the calcium-sensing receptor (calcilytics) stimulate PTH secretion and represent thus an alternative approach to elevate hormone levels transiently. We report here on ATF936, a novel calcilytic, which triggered rapid, transient spikes in endogenous PTH levels when given orally in single doses of 10 and 30mg/kg to growing rats, and of 1mg/kg to dogs. Eight weeks daily oral application of 30mg/kg of ATF936 to aged female rats induced in the proximal tibia metaphysis increases in bone mineral density, cancellous bone volume and cortical and trabecular thickness as evaluated by computed tomography. In healthy humans, single oral doses of ATF936 produced peak PTH levels in plasma after a median time of 1h and levels returned to normal at 24-h post-dose. The average maximum PTH concentration increase from baseline was 1.9, 3.6, and 6.0-fold at doses of 40, 70, and 140mg. ATF936 was well tolerated. The sharp, transient increase in PTH levels produced by the oral calcilytic ATF936 was comparable to the PTH profile observed after subcutaneous administration of teriparatide. In conclusion, ATF936 might hold potential as an oral bone-forming osteoporosis therapy.

Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/β-catenin signaling. We found the extracellular β-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.

Beta-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific beta-catenin-deficient mice (Ctnnb1(loxP/loxP); Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/beta-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific beta-catenin mutants. Together, these results reveal a crucial novel function for osteocyte beta-catenin signaling in controlling bone homeostasis.

Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.

Achondroplasia (ACH) is the most frequent form of dwarfism and is caused by gain-of-function mutations in the fibroblast growth factor receptor 3-encoding (FGFR3-encoding) gene. Although potential therapeutic strategies for ACH, which aim to reduce excessive FGFR3 activation, have emerged over many years, the use of tyrosine kinase inhibitor (TKI) to counteract FGFR3 hyperactivity has yet to be evaluated. Here, we have reported that the pan-FGFR TKI, NVP-BGJ398, reduces FGFR3 phosphorylation and corrects the abnormal femoral growth plate and calvaria in organ cultures from embryos of the Fgfr3Y367C/+ mouse model of ACH. Moreover, we demonstrated that a low dose of NVP-BGJ398, injected subcutaneously, was able to penetrate into the growth plate of Fgfr3Y367C/+ mice and modify its organization. Improvements to the axial and appendicular skeletons were noticeable after 10 days of treatment and were more extensive after 15 days of treatment that started from postnatal day 1. Low-dose NVP-BGJ398 treatment reduced intervertebral disc defects of lumbar vertebrae, loss of synchondroses, and foramen-magnum shape anomalies. NVP-BGJ398 inhibited FGFR3 downstream signaling pathways, including MAPK, SOX9, STAT1, and PLCγ, in the growth plates of Fgfr3Y367C/+ mice and in cultured chondrocyte models of ACH. Together, our data demonstrate that NVP-BGJ398 corrects pathological hallmarks of ACH and support TKIs as a potential therapeutic approach for ACH.

We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4-agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.

Intermittent parathyroid hormone (PTH) application is an established pharmacological principle to stimulate bone formation. Yet, the molecular mechanisms underlying this bone anabolic action are not fully understood. Recently, SOST (sclerostin) was identified as a potent osteocyte expressed negative regulator of bone formation in vitro, in murine models and in patients with the bone overgrowth disorders Sclerosteosis and Van Buchem disease. Therefore, we have studied the impact of PTH on SOST regulation. First, we analyzed SOST expression during PTH-induced bone formation in a classical model of local bone formation. 8-month-old mice received intermittently 100 nM hPTH(1–34) or vehicle onto the calvaria for 5 days. PTH stimulated bone formation as assessed by fluorochrome-marker-based histomorphometry. SOST expression was reduced in PTH-treated calvariae 4 h after the last administration as evaluated by real-time quantitative PCR. Next, we observed a decrease of SOST expression in femoral cortical bone of 6-month-old rats following single subcutaneous systemic administration of 80 μg/kg PTH(1–34). Finally, we studied SOST mRNA expression in bone of 11-month-old osteopenic estrogen-deprived (OVX) rats following 8-week systemic intermittent administration of 5 μg/kg PTH(1–34). PTH-treated animals displayed increases in bone mineral density as detected by pQCT, while SOST mRNA levels were decreased compared to vehicle-treated OVX and SHAM controls. PTH decreased SOST expression also in vitro. 100 nM PTH(1–34) inhibited expression in mouse calvaria organ cultures and in osteoblastic UMR-106 cells within 6 h by 95%. An IC 50 of 1 nM was determined for PTH(1–34) in UMR-106 cells, whereas the PTH antagonist ( d -Trp 12 ,Tyr 34 )-bPTH(7–34) did not efficiently reduce SOST expression. Furthermore, SOST inhibition by PTH was not blocked by the protein synthesis inhibitor cycloheximide, indicating direct regulation, and PTH did not influence SOST mRNA degradation, implying transcriptional regulation. Finally, we observed full suppression of SOST by the cAMP inducer forskolin, partial inhibition by ionomycin, and no effect with PMA, indicating that PTH action is mainly mediated via the cAMP/PKA pathway. In summary, we have shown that PTH directly inhibits SOST transcription in vivo and in vitro, suggesting that SOST regulation may play a role in mediating PTH action in bone.

Thiazolidinediones are insulin-sensitizing agents and in clinical use for the treatment of type II diabetes. Under specific experimental conditions, these molecules induce adipogenic differentiation of mesenchymal precursor cells at the expense of osteoblasts in vitro, suggesting possible negative effects on the skeleton. We measured effects of the thiazolidinedione BRL49653 on bone tissue of intact and estrogen-deprived skeletally mature adult female Wistar rats (6-9 months old). Weight gain and decreased plasma triglyceride levels confirmed the effectiveness of the treatment. However, no change in bone mass or fat marrow volume was observed in intact rats treated for 8 weeks with 5, 10, or 20 mg/kg of BRL49653. Study of marrow cultures established at necropsy revealed a higher responsiveness to adipogenic differentiation protocols of cultures established from the 10-mg/kg group compared to vehicle control. In a second study, the effects of thiazolidinedione treatment on the skeleton of estrogen-deprived rats were investigated. Application of 10 mg/kg of BRL49653 for 12 weeks resulted in enhanced bone loss (+31%; pQCT) and increased fat marrow volume (+117%; histomorphometry) compared to vehicle-treated OVX control. Interestingly, osteoblast number was comparable in both cases. Bone resorption parameters were significantly increased in the treatment group (+27% osteoclast number, +30% eroded surface). Enhanced bone loss due to treatment was consistently observed in the tibia, femur, and the lumbar spine. Our data indicate that thiazolidinediones may enhance bone loss induced by estrogen deprivation.

Objectives: Soft and hard tissue healing around submerged and nonsubmerged versions of one dental implant design was evaluated in an experimental canine peri-implantitis model. Material and methods: Forty-eight c.p. Ti-implants with integrated (one-piece=OPI) or screw-on (two-piece=TPI) abutments were inserted in edentulous mandibles of eight beagle dogs, one OPI and one TPI with connected abutments for nonsubmerged and one TPI without abutment (SMI) for submerged healing. After 3 months, all implants were functionally loaded, and at 4 months peri-implantitis was ligature-induced in one jaw side. Intravital polyfluorochrome labeling, monthly conventional radiography and gingival probing of all 48 implants were performed until sacrifice 8 months postimplantation. Undecalcified ground sections in the bucco-lingual and mesio-distal planes of four dogs (23 implants, one implant lost) were evaluated by light and fluorescence microscopy. The immunohistochemical and SEM-vascular corrosion cast results of the four other dogs (24 implants) will be reported elsewhere. Levels of alveolar bone-to-implant contact (ABICL), alveolar crest (ACL) and junctional epithelium-to-implant contact were determined by computer-assisted histometry. Peri-implant alveolar bone loss (=saucerization) was assessed on the radiographs and calculated as ACL minus ABICL from histometric data. Results: Around SMIs and OPIs without ligature less plaque adhesion and lower gingival indices were found when compared to TPIs. Radiologically, all ligatured, but also some nonligatured implants showed alveolar bone loss. Histometry demonstrated reduced ABICL around all these implants. Saucerization was more pronounced on the lingual and mesio-distal sides. Particularly around TPIs, bone resorption was still active or bone formation was impaired on fluorochrome labeling. Only around SMIs and one OPI without ligature continuing alveolar bone formation reflected by gains in ABICL were found. Conclusion: The clinical and histometric results of this study demonstrate that healing of submerged SMIs was not impaired by the two-stage procedure, resulting in equally good healing as around nonsubmerged OPIs. However, peri-implantitis plaque-induced by ligature and/or dilated abutment connection microgaps in TPIs affected alveolar bone-to-implant contacts more than transmucosal or submerged healing mechanisms.

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost(-/-) mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14days), as well as at 28days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area. Normal endochondral ossification progressed in wild type and Sost(-/-) mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost(-/-) fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost(-/-) calluses over wild type with greater bone volume at day 10 (221%, p