RÉSUMÉ
Pathogenic variants disrupting the binding between sclerostin (encoded by SOST) and its receptor LRP4 have previously been described to cause sclerosteosis, a rare high bone mass disorder. The sclerostin-LRP4 complex inhibits canonical WNT signaling, a key pathway regulating osteoblastic bone formation and a promising therapeutic target for common bone disorders, such as osteoporosis. In the current study, we crossed mice deficient for Sost (Sost-/-) with our p.Arg1170Gln Lrp4 knock-in (Lrp4KI/KI) mouse model to create double mutant Sost-/-;Lrp4KI/KI mice. We compared the phenotype of Sost-/- mice with that of Sost-/-;Lrp4KI/KI mice, to investigate a possible synergistic effect of the disease-causing p.Arg1170Trp variant in Lrp4 on Sost deficiency. Interestingly, presence of Lrp4KI alleles partially mitigated the Sost-/- phenotype. Cellular and dynamic histomorphometry did not reveal mechanistic insights into the observed phenotypic differences. We therefore determined the molecular effect of the Lrp4KI allele by performing bulk RNA sequencing on Lrp4KI/KI primary osteoblasts. Unexpectedly, mostly genes related to bone resorption or remodeling (Acp5, Rankl, Mmp9) were upregulated in Lrp4KI/KI primary osteoblasts. Verification of these markers in Lrp4KI/KI, Sost-/- and Sost-/-;Lrp4KI/KI mice revealed that sclerostin deficiency counteracts this Lrp4KI/KI effect in Sost-/-;Lrp4KI/KI mice. We therefore hypothesize that models with two inactivating Lrp4KI alleles rather activate bone remodeling, with a net gain in bone mass, whereas sclerostin deficiency has more robust anabolic effects on bone formation. Moreover, these effects of sclerostin and Lrp4 are stronger in female mice, contributing to a more severe phenotype than in males and more detectable phenotypic differences among different genotypes.
Sujet(s)
Protéines adaptatrices de la transduction du signal , Remodelage osseux , Hyperostose , Syndactylie , Mâle , Femelle , Animaux , Souris , Protéines adaptatrices de la transduction du signal/génétique , Protéines adaptatrices de la transduction du signal/métabolisme , Souris knockout , Phénotype , Mutation , Remodelage osseux/génétique , Protéines apparentées au récepteur LDL/génétique , Protéines apparentées au récepteur LDL/métabolismeRÉSUMÉ
Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects 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 forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including 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), at 3 months of age 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 subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.
Sujet(s)
Mutation gain de fonction , Hyperostose , Animaux , Humains , Souris , Protéines adaptatrices de la transduction du signal , Sécrétions corporelles , Modèles animaux de maladie humaine , Hyperostose/génétique , Protéine-5 apparentée au récepteur des LDL/génétique , MutationRÉSUMÉ
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 α5ß1 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.
Sujet(s)
Cartilage articulaire , Gonarthrose , Humains , Gonarthrose/traitement médicamenteux , Chondrocytes , Transduction du signal , Angiopoïétines/métabolisme , Angiopoïétines/pharmacologie , Angiopoïétines/usage thérapeutique , Protéine-3 de type angiopoïétineRÉSUMÉ
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 µÎµ 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 µÎµ 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.
Sujet(s)
Protéines adaptatrices de la transduction du signal/génétique , Hyperostose/génétique , Ostéogenèse/génétique , Contrainte mécanique , Syndactylie/génétique , Animaux , Résorption osseuse/génétique , Résorption osseuse/physiopathologie , Os et tissu osseux/physiopathologie , Os cortical/physiologie , Femelle , Glycoprotéines/génétique , Hyperostose/physiopathologie , Mâle , Souris , Souris knockout , Ostéogenèse/physiologie , Syndactylie/physiopathologieRÉSUMÉ
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.
Sujet(s)
Résorption osseuse/métabolisme , Os et tissu osseux/métabolisme , Os spongieux/métabolisme , Ostéoporose/métabolisme , Temps , Protéines adaptatrices de la transduction du signal/métabolisme , Animaux , Femelle , Glycoprotéines/métabolisme , Mâle , Souris , Ostéogenèse/effets des médicaments et des substances chimiques , Ostéogenèse/physiologie , Microtomographie aux rayons X/méthodesRÉSUMÉ
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.
Sujet(s)
Sous-pesanteur , Protéines du muscle/déficit , Muscles squelettiques/métabolisme , Muscles squelettiques/anatomopathologie , Amyotrophie/étiologie , Amyotrophie/métabolisme , Vol spatial , Protéines à motif tripartite/déficit , Ubiquitin-protein ligases/déficit , Animaux , Prédisposition aux maladies , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes , Suspension des membres postérieurs , Souris , Souris knockout , Amyotrophie/anatomopathologie , Taille d'organeRÉSUMÉ
Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/ß-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 < 0.01; + 22% wild type p < 0.05). Scl-Ab treatment produced less marked increases in cortical bone of the tibiae, with anabolic effects similar across genotypes. Mechanical testing confirmed that Scl-Ab improved strength across all genotypes; however, no enhancement was seen within Dkk1 KO mice. Dynamic bone labeling showed that Scl-Ab treatment was associated with increased bone formation, regardless of genotype. Immunohistochemical staining for sclerostin protein indicated no differences in the Dkk1 KO mice, indicating that the increased Wnt signaling associated with DKK1 deficiency was not compensated by upregulation of sclerostin protein. These data suggest complex interactions between Wnt signaling factors in bone, but critically illustrate synergy between DKK1 deficiency and Scl-Ab treatment. These data support the application of dual-targeted therapeutics in the modulation of bone anabolism.
Sujet(s)
Os et tissu osseux/effets des médicaments et des substances chimiques , Os et tissu osseux/physiologie , Glycoprotéines/métabolisme , Protéines et peptides de signalisation intercellulaire/métabolisme , Ostéogenèse/physiologie , Protéines adaptatrices de la transduction du signal , Animaux , Anticorps monoclonaux/pharmacologie , Femelle , Souris , Souris knockout , Ostéogenèse/effets des médicaments et des substances chimiquesRÉSUMÉ
Sclerostin, a product of the Sost gene, is a Wnt-inhibitor and thus negatively regulates bone accrual. Canonical Wnt/ß-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.
Sujet(s)
Glycoprotéines/déficit , Tibia/croissance et développement , Protéines adaptatrices de la transduction du signal , Animaux , Densité osseuse , Développement osseux/génétique , Analyse des éléments finis , Glycoprotéines/génétique , Protéines et peptides de signalisation intercellulaire , Souris , Souris knockout , Contrainte mécaniqueRÉSUMÉ
Hydrogels are under active development for controlled drug delivery, but their clinical translation is 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-1 ) over four weeks. The release rate is primarily governed by ionic exchange and can be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury is 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.
Sujet(s)
Tendon calcanéen , Matériaux biomimétiques , Vecteurs de médicaments , Hydrogels , Facteur de croissance IGF-I , Peptides , Traumatismes des tendons , Tendon calcanéen/métabolisme , Tendon calcanéen/anatomopathologie , Animaux , Matériaux biomimétiques/composition chimique , Matériaux biomimétiques/pharmacocinétique , Matériaux biomimétiques/pharmacologie , Préparations à action retardée/composition chimique , Préparations à action retardée/pharmacocinétique , Préparations à action retardée/pharmacologie , Vecteurs de médicaments/composition chimique , Vecteurs de médicaments/pharmacocinétique , Vecteurs de médicaments/pharmacologie , Femelle , Humains , Hydrogels/composition chimique , Hydrogels/pharmacocinétique , Hydrogels/pharmacologie , Souris , Cellules NIH 3T3 , Peptides/composition chimique , Peptides/pharmacocinétique , Peptides/pharmacologie , Rats , Rat Sprague-Dawley , Traumatismes des tendons/traitement médicamenteux , Traumatismes des tendons/métabolisme , Traumatismes des tendons/anatomopathologieRÉSUMÉ
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.
Sujet(s)
Adipocytes/métabolisme , Adipogenèse , Tissu adipeux/métabolisme , Cellules de la moelle osseuse/métabolisme , Glycoprotéines/métabolisme , Cellules souches mésenchymateuses/métabolisme , Ostéocytes/métabolisme , Cellules 3T3-L1 , Protéines adaptatrices de la transduction du signal , Tissu adipeux/cytologie , Adiposité , Animaux , Milieux de culture conditionnés/métabolisme , Glycoprotéines/déficit , Glycoprotéines/génétique , Protéines et peptides de signalisation intercellulaire , Mâle , Souris , Souris de lignée C57BL , Souris knockout , Communication paracrine , Phénotype , Niche de cellules souches , Voie de signalisation WntRÉSUMÉ
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 11weeks. 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.
Sujet(s)
Ostéodystrophie rénale/métabolisme , Ostéodystrophie rénale/anatomopathologie , Glycoprotéines/déficit , Protéines adaptatrices de la transduction du signal , Animaux , Femelle , Protéines et peptides de signalisation intercellulaire , Souris , Souris de lignée C57BL , Souris knockoutRÉSUMÉ
OBJECTIVE: To establish an unbiased, 3-dimensional (3-D) approach that quantifies subchondral bone plate (SBP) changes in mouse joints, and to investigate the mechanism that mediates SBP sclerosis at a late stage of osteoarthritis (OA). METHODS: A new micro-computed tomography (micro-CT) protocol was developed to characterize the entire thickness of the SBP in the distal femur of a normal mouse knee. Four mouse models of severe joint OA were generated: cartilage-specific Egfr-knockout (Egfr-CKO) mice at 2 months after surgical destabilization of the medial meniscus (DMM), Egfr-CKO mice with aging-related spontaneous OA, wild-type (WT) mice at 10 months after DMM, and WT mice at 14 weeks after DMM plus hemisectomy of the meniscus (DMMH) surgery. As an additional model, mice with knockout of the sclerostin gene (Sost-KO) were subjected to DMMH surgery. Knee joints were examined by micro-CT, histology, and immunohistochemical analyses. RESULTS: Examination of the mouse distal femur by 3-D micro-CT revealed a positive correlation between SBP thickness and the loading status in normal knees. In all 4 mouse models of late-stage OA, SBP sclerosis was restricted to the areas under severely eroded articular cartilage. This was accompanied by elevated bone formation at the bone marrow side of the SBP and a drastic reduction in the levels of sclerostin in osteocytes within the SBP. Unlike in WT mice, no further increase in the thickness of the SBP was observed in response to DMMH in Sost-KO mice. CONCLUSION: Since focal stress on the SBP underlying sites of cartilage damage increases during late stages of OA, these findings establish mechanical loading-induced attenuation of sclerostin expression and elevation of bone formation along the SBP surface as the major mechanisms characterizing subchondral bone phenotypes associated with severe late-stage OA in mice.
Sujet(s)
Os et tissu osseux/anatomopathologie , Glycoprotéines/métabolisme , Articulation du genou/anatomopathologie , Gonarthrose/anatomopathologie , Ostéosclérose/étiologie , Protéines adaptatrices de la transduction du signal , Animaux , Os et tissu osseux/métabolisme , Modèles animaux de maladie humaine , Fémur/anatomopathologie , Immunohistochimie , Protéines et peptides de signalisation intercellulaire , Articulation du genou/métabolisme , Mâle , Souris , Souris knockout , Gonarthrose/complications , Gonarthrose/métabolisme , Ostéosclérose/métabolisme , Contrainte mécanique , Microtomographie aux rayons XRÉSUMÉ
Decreased activity or expression of sclerostin, an endogenous inhibitor of Wnt/ß-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/ß-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/ß-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.
Sujet(s)
Anticorps/pharmacologie , Glycoprotéines/immunologie , Ostéogenèse/effets des médicaments et des substances chimiques , Tibia/physiologie , Protéines adaptatrices de la transduction du signal , Animaux , Densité osseuse/effets des médicaments et des substances chimiques , Régulation négative/effets des médicaments et des substances chimiques , Régulation négative/génétique , Femelle , Humains , Protéines et peptides de signalisation intercellulaire , Souris de lignée C57BL , Modèles animaux , Taille d'organe/effets des médicaments et des substances chimiques , Transduction du signal/effets des médicaments et des substances chimiques , Transduction du signal/génétique , Tibia/anatomie et histologie , Tibia/imagerie diagnostique , Tibia/effets des médicaments et des substances chimiques , Mise en charge/physiologie , Microtomographie aux rayons XRÉSUMÉ
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.
Sujet(s)
Os cortical/métabolisme , Régulation de l'expression des gènes , Glycoprotéines/déficit , Ostéogenèse/génétique , Contrainte mécanique , Protéines adaptatrices de la transduction du signal , Analyse de variance , Animaux , Calcification physiologique , Os cortical/imagerie diagnostique , Os cortical/croissance et développement , Femelle , Protéines et peptides de signalisation intercellulaire , Mâle , Souris , Souris knockout , Modèles animaux , Microtomographie aux rayons XRÉSUMÉ
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.
Sujet(s)
Densité osseuse/effets des médicaments et des substances chimiques , Protéines morphogénétiques osseuses/antagonistes et inhibiteurs , Fractures osseuses/prévention et contrôle , Ostéocytes/composition chimique , Ostéogenèse/effets des médicaments et des substances chimiques , Protéines adaptatrices de la transduction du signal , Animaux , Anticorps/pharmacologie , Anticorps/usage thérapeutique , Protéines morphogénétiques osseuses/immunologie , Lignée cellulaire tumorale , Diphosphonates/usage thérapeutique , Marqueurs génétiques/immunologie , Humains , Imidazoles/usage thérapeutique , Souris , Myélome multiple/complications , Cellules cancéreuses en culture , Acide zolédroniqueRÉSUMÉ
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, P<0.05). 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.
Sujet(s)
Anticorps/usage thérapeutique , Diphosphonates/usage thérapeutique , Glycoprotéines/immunologie , Imidazoles/usage thérapeutique , Ostéogenèse imparfaite/traitement médicamenteux , Protéines adaptatrices de la transduction du signal , Animaux , Anticorps/immunologie , Densité osseuse/effets des médicaments et des substances chimiques , Densité osseuse/physiologie , Modèles animaux de maladie humaine , Femelle , Glycoprotéines/antagonistes et inhibiteurs , Protéines et peptides de signalisation intercellulaire , Souris , Ostéogenèse/effets des médicaments et des substances chimiques , Ostéogenèse/physiologie , Ostéogenèse imparfaite/physiopathologie , Acide zolédroniqueRÉSUMÉ
Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/ß-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/ß-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.
Sujet(s)
Résorption osseuse/étiologie , Résorption osseuse/métabolisme , Glycoprotéines/métabolisme , Ostéoblastes/métabolisme , Lésions radiques/complications , Lésions radiques/métabolisme , Cellules souches/métabolisme , Protéines adaptatrices de la transduction du signal , Animaux , Anticorps/pharmacologie , Anticorps/usage thérapeutique , Apoptose/effets des médicaments et des substances chimiques , Moelle osseuse/anatomopathologie , Os spongieux/anatomopathologie , Lignage cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Réparation de l'ADN/effets des médicaments et des substances chimiques , Protéines et peptides de signalisation intercellulaire , Mâle , Mésoderme/anatomopathologie , Souris de lignée C57BL , Ostéoblastes/effets des médicaments et des substances chimiques , Ostéocytes/effets des médicaments et des substances chimiques , Ostéocytes/anatomopathologie , Ostéogenèse/effets des médicaments et des substances chimiques , Cellules souches/effets des médicaments et des substances chimiquesRÉSUMÉ
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.
Sujet(s)
Densité osseuse , Ostéodystrophie rénale/étiologie , Glycoprotéines/génétique , Ostéogenèse , Insuffisance rénale chronique/complications , Protéines adaptatrices de la transduction du signal , Animaux , Os et tissu osseux/cytologie , Os et tissu osseux/anatomopathologie , Ostéodystrophie rénale/génétique , Ostéodystrophie rénale/métabolisme , Modèles animaux de maladie humaine , Femelle , Génotype , Glycoprotéines/métabolisme , Protéines et peptides de signalisation intercellulaire , Souris , Souris de lignée C57BL , Souris knockout , Ostéoblastes/anatomopathologie , Insuffisance rénale chronique/métabolisme , Tibia , Microtomographie aux rayons XRÉSUMÉ
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.
Sujet(s)
Achondroplasie/génétique , Cartilage/malformations , Mandibule/malformations , Condyle mandibulaire/malformations , Récepteur de type 3 des facteurs de croissance fibroblastique/génétique , Achondroplasie/imagerie diagnostique , Achondroplasie/traitement médicamenteux , Achondroplasie/physiopathologie , Animaux , Cartilage/croissance et développement , Cartilage/physiopathologie , Différenciation cellulaire/effets des médicaments et des substances chimiques , Différenciation cellulaire/génétique , Prolifération cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/génétique , Chondrocytes/métabolisme , Chondrocytes/anatomopathologie , Modèles animaux de maladie humaine , Humains , Mandibule/croissance et développement , Mandibule/physiopathologie , Condyle mandibulaire/croissance et développement , Condyle mandibulaire/physiopathologie , Souris , Ostéogenèse/effets des médicaments et des substances chimiques , Ostéogenèse/génétique , Phénylurées/administration et posologie , Inhibiteurs de protéines kinases/administration et posologie , Pyrimidines/administration et posologieRÉSUMÉ
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.