RESUMEN
OBJECTIVE: IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. METHODS: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n = 3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n = 11-13). RESULTS: Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). CONCLUSION: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.
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Proteínas del Linfoma 3 de Células B , Huesos , Osteogénesis , Animales , Ratones , Huesos/metabolismo , Densidad Ósea , Diferenciación Celular , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteínas del Linfoma 3 de Células B/genéticaRESUMEN
The osteogenesis imperfecta murine (oim) model with solely homotrimeric (α1)3 type I collagen, owing to a dysfunctional α2(I) collagen chain, has a brittle bone phenotype, implying that the (α1)2(α2)1 heterotrimer is required for physiological bone function. Here, we comprehensively show, for the first time, that mice lacking the α2(I) chain do not have impaired bone biomechanical or structural properties, unlike oim homozygous mice. However, Mendelian inheritance was affected in male mice of both lines, and male mice null for the α2(I) chain exhibited age-related loss of condition. Compound heterozygotes were generated to test whether gene dosage was responsible for the less-severe phenotype of oim heterozygotes, after allelic discrimination showed that the oim mutant allele was not downregulated in heterozygotes. Compound heterozygotes had impaired bone structural properties compared to those of oim heterozygotes, albeit to a lesser extent than those of oim homozygotes. Hence, the presence of heterotrimeric type I collagen in oim heterozygotes alleviates the effect of the oim mutant allele, but a genetic interaction between homotrimeric type I collagen and the oim mutant allele leads to bone fragility.
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Osteogénesis Imperfecta , Animales , Colágeno/genética , Colágeno Tipo I/genética , Modelos Animales de Enfermedad , Homocigoto , Masculino , Ratones , Ratones Mutantes , Osteogénesis Imperfecta/genéticaRESUMEN
Paget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml-/- mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml-/- was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml-/- mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml-/- mice and wild type, bone histomorphometry showed that Pml-/- mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper.
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Resorción Ósea , Osteítis Deformante , Animales , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Osteítis Deformante/genética , Osteoclastos/metabolismo , Osteogénesis , Proteína de la Leucemia PromielocíticaRESUMEN
Short stature and osteoporosis are common in Duchenne muscular dystrophy (DMD) and its pathophysiology may include an abnormality of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis, which is further exacerbated by long-term glucocorticoid (GC) treatment. Hence, an agent that has anabolic properties and may improve linear growth would be beneficial in this setting and therefore requires further exploration. A 5-week-old x-linked muscular dystrophy (mdx) mice were used as a model of DMD. They were treated with prednisolone ± GH + IGF-1 for 4 weeks and then compared to control mdx mice to allow the study of both growth and skeletal structure. GC reduced cortical bone area, bone fraction, tissue area and volume and cortical bone volume, as assessed by micro computed tomography (CT) In addition, GC caused somatic and skeletal growth retardation but improved grip strength. The addition of GH + IGF-1 therapy rescued the somatic growth retardation and induced additional improvements in grip strength (16.9% increase, P < 0.05 compared to control). There was no improvement in bone microarchitecture (assessed by micro-CT and static histomorphometry) or biomechanical properties (assessed by three-point bending). Serum bone turnover markers (Serum procollagen 1 intact N-terminal propeptide (P1NP), alpha C-terminal telopeptide (αCTX)) also remained unaffected. Further work is needed to maximise these gains before proceeding to clinical trials in boys with DMD.
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Enfermedades Óseas Metabólicas , Hormona de Crecimiento Humana , Distrofia Muscular de Duchenne , Animales , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Enfermedades Óseas Metabólicas/etiología , Enfermedades Óseas Metabólicas/prevención & control , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Trastornos del Crecimiento/tratamiento farmacológico , Trastornos del Crecimiento/prevención & control , Hormona del Crecimiento/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/tratamiento farmacológico , Microtomografía por Rayos XRESUMEN
Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.
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Artritis Reumatoide/genética , Proteínas de Drosophila/genética , Músculo Esquelético/metabolismo , Receptor Smoothened/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Cartílago/crecimiento & desarrollo , Cartílago/metabolismo , Cartílago/patología , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Proteínas Hedgehog/genética , Homeostasis/genética , Humanos , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Ratones , Músculo Esquelético/patología , Osteogénesis/genética , Transducción de Señal/genética , Tendones/metabolismo , Tendones/patologíaRESUMEN
Early onset familial Paget's disease of bone (EoPDB), familial expansile osteolysis, and expansile skeletal hyperphosphatasia are related disorders caused by insertion mutations in exon 1 of the TNFRSF11A gene, which encodes receptor activator of nuclear factor κB (RANK) protein. To understand the mechanisms underlying these disorders, we developed a mouse model carrying the 75dup27 mutation which causes EoPDB. Mice heterozygous for the mutation (Tnfrsf11a75dup27/- ) developed a PDB-like disorder with focal osteolytic lesions in the hind limbs with increasing age. Treatment of these mice with zoledronic acid completely prevented the development of lesions. Studies in vitro showed that RANK ligand (RANKL)-induced osteoclast formation and signaling was impaired in bone marrow cells from Tnfrsf11a75dup27/- animals, but that osteoclast survival was increased independent of RANKL stimulation. Surprisingly, Tnfrsf11a75dup27/75dup27 homozygotes had osteopetrosis at birth, with complete absence of osteoclasts. Bone marrow cells from these mice failed to form osteoclasts in response to RANKL and macrophage colony-stimulating factor (M-CSF) stimulation. This intriguing study has shown that in heterozygous form, the 75dup27 mutation causes focal osteolytic lesions in vivo reminiscent of the human disorder and extends osteoclast survival independently of RANKL signaling. In homozygous form, however, the mutation causes osteopetrosis due to failure of osteoclast formation and insensitivity to RANKL stimulation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..
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Osteítis Deformante , Osteopetrosis , Animales , Homocigoto , Humanos , Ratones , Mutagénesis Insercional , Mutación , Osteítis Deformante/genética , Osteoclastos , Osteopetrosis/genética , Fenotipo , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
BACKGROUND: Osteoarthritis (OA) is a whole joint disease that affects all joint tissues, with changes in the articular cartilage (AC), subchondral bone and synovium. Pathologies in menisci and ligaments, however, are rarely analysed, although both are known to play vital roles in the mechanical stability of the joint. The aim of our study was to describe the pathological changes in menisci and ligament during disease development in murine spontaneous and post-traumatic surgically induced OA and to quantify tissue mineralisation in the joint space using micro-computed tomography (µCT) imaging during OA progression. METHODS: Knees of Str/ort mice (spontaneous OA model; 26-40 weeks) and C57CBA F1 mice following destabilisation of medial meniscus (DMM) surgery (post-traumatic OA model; 8 weeks after DMM), were used to assess histological meniscal and ligament pathologies. Joint space mineralised tissue volume was quantified by µCT. RESULTS: Meniscal pathological changes in Str/ort mouse knees were associated with articular cartilage lesion severity. These meniscal changes included ossification, hyperplasia, cell hypertrophy, collagen type II deposition and Sox9 expression in the fibrous region near the attachment to the knee joint capsule. Anterior cruciate ligaments exhibited extracellular matrix changes and chondrogenesis particularly at the tibial attachment site, and ossification was seen in collateral ligaments. Similar changes were confirmed in the post-traumatic DMM model. µCT analysis showed increased joint space mineralised tissue volume with OA progression in both the post-traumatic and spontaneous OA models. CONCLUSIONS: Modifications in meniscal and ligament mineralisation and chondrogenesis are seen with overt AC degeneration in murine OA. Although the aetiology and the consequences of such changes remain unknown, they will influence stability and load transmission of the joint and may therefore contribute to OA progression. In addition, these changes may have important roles in movement restriction and pain, which represent major human clinical symptoms of OA. Description of such soft tissue changes, in addition to AC degradation, should be an important aspect of future studies in mouse models in order to furnish a more complete understanding of OA pathogenesis.
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Cartílago Articular , Osteoartritis , Animales , Ligamento Cruzado Anterior , Cartílago Articular/diagnóstico por imagen , Modelos Animales de Enfermedad , Meniscos Tibiales/diagnóstico por imagen , Ratones , Osteoartritis/diagnóstico por imagen , Osteoartritis/etiología , Microtomografía por Rayos XRESUMEN
Lactation-induced bone loss occurs due to high calcium requirements for fetal growth but skeletal recovery is normally achieved promptly postweaning. Dietary protein is vital for fetus and mother but the effects of protein undernutrition on the maternal skeleton and skeletal muscles are largely unknown. We used mouse dams fed with normal (N, 20%) or low (L, 8%) protein diet during gestation and lactation and maintained on the same diets (NN, LL) or switched from low to normal (LN) during a 28 d skeletal restoration period post lactation. Skeletal muscle morphology and neuromuscular junction integrity was not different between any of the groups. However, dams fed the low protein diet showed extensive bone loss by the end of lactation, followed by full skeletal recovery in NN dams, partial recovery in LN and poor bone recovery in LL dams. Primary osteoblasts from low protein diet fed mice showed decreased in vitro bone formation and decreased osteogenic marker gene expression; promoter methylation analysis by pyrosequencing showed no differences in Bmpr1a, Ptch1, Sirt1, Osx, and Igf1r osteoregulators, while miR-26a, -34a, and -125b expression was found altered in low protein fed mice. Therefore, normal protein diet is indispensable for maternal musculoskeletal health during the reproductive period.
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Fenómenos Fisiológicos Nutricionales de los Animales/fisiología , Enfermedades Óseas Metabólicas/fisiopatología , Dieta con Restricción de Proteínas , Lactancia/fisiología , Músculo Esquelético/fisiología , Reproducción/fisiología , Animales , Animales Recién Nacidos , Peso Corporal , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones Transgénicos , MicroARNs/genética , Músculo Esquelético/metabolismo , Osteoblastos/metabolismo , Osteogénesis/genética , DesteteRESUMEN
The muscular dystrophy X-linked (mdx) mouse is commonly used as a mouse model of Duchenne muscular dystrophy (DMD). Its phenotype is, however, mild, and other mouse models have been explored. The mdx:Cmah-/- mouse carries a human-like mutation in the Cmah gene and has a severe muscle phenotype, but its growth and bone development are unknown. In this study, we compared male mdx, mdx:Utrn+/-, mdx:Cmah-/- and wild-type (WT) mice at 3, 5 and 7â weeks of age to determine the suitability of the mdx:Cmah-/- mouse as a model for assessing growth and skeletal development in DMD. The mdx:Cmah-/- mice were lighter than WT mice at 3 weeks, but heavier at 7â weeks, and showed an increased growth rate at 5â weeks. Cortical bone fraction as assessed by micro-computed tomography was greater in both mdx and mdx:Cmah-/- mice versus WT mice at 7â weeks. Tissue mineral density was also higher in mdx:Cmah-/- mice at 3 and 7 weeks. Gene profiling of mdx:Cmah-/- bone identified increased expression of Igf1, Igf1r and Vegfa Both the mdx and mdx:Cmah-/- mice showed an increased proportion of regulated bone marrow adipose tissue (BMAT) but a reduction in constitutive BMAT. The mdx:Cmah-/- mice show evidence of catch-up growth and more rapid bone development. This pattern does not mimic the typical DMD growth trajectory and therefore the utility of the mdx:Cmah-/- mouse for studying growth and skeletal development in DMD is limited. Further studies of this model may, however, shed light on the phenomenon of catch-up growth.This article has an associated First Person interview with the first author of the paper.
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Desarrollo Óseo , Huesos/patología , Utrofina/metabolismo , Adiposidad , Animales , Fenómenos Biomecánicos , Médula Ósea/patología , Huesos/diagnóstico por imagen , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/patología , Hueso Esponjoso/fisiopatología , Hueso Cortical/diagnóstico por imagen , Hueso Cortical/patología , Hueso Cortical/fisiopatología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Placa de Crecimiento/diagnóstico por imagen , Placa de Crecimiento/patología , Placa de Crecimiento/fisiopatología , Fuerza de la Mano , Inflamación/patología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/diagnóstico por imagen , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tibia/diagnóstico por imagen , Tibia/patología , Tibia/fisiopatología , Microtomografía por Rayos XRESUMEN
Paget's disease of bone (PDB) is a chronic skeletal disorder that can affect one or several bones in individuals older than 55 y of age. PDB-like changes have been reported in archaeological remains as old as Roman, although accurate diagnosis and natural history of the disease is lacking. Six skeletons from a collection of 130 excavated at Norton Priory in the North West of England, which dates to medieval times, show atypical and extensive pathological changes resembling contemporary PDB affecting as many as 75% of individual skeletons. Disease prevalence in the remaining collection is high, at least 16% of adults, with age at death estimations as low as 35 y. Despite these atypical features, paleoproteomic analysis identified sequestosome 1 (SQSTM1) or p62, a protein central to the pathological milieu of PDB, as one of the few noncollagenous human sequences preserved in skeletal samples. Targeted proteomic analysis detected >60% of the ancient p62 primary sequence, with Western blotting indicating p62 abnormalities, including in dentition. Direct sequencing of ancient DNA excluded contemporary PDB-associated SQSTM1 mutations. Our observations indicate that the ancient p62 protein is likely modified within its C-terminal ubiquitin-associated domain. Ancient miRNAs were remarkably preserved in an osteosarcoma from a skeleton with extensive disease, with miR-16 expression consistent with that reported in contemporary PDB-associated bone tumors. Our work displays the use of proteomics to inform diagnosis of ancient diseases such as atypical PDB, which has unusual features presumably potentiated by yet-unidentified environmental or genetic factors.
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Huesos/metabolismo , Osteítis Deformante/metabolismo , Proteoma , Proteína Sequestosoma-1/metabolismo , Huesos/patología , Historia Medieval , Humanos , MicroARNs/metabolismo , Osteítis Deformante/complicaciones , Osteítis Deformante/patología , Osteosarcoma/etiología , Osteosarcoma/metabolismo , Paleopatología , Análisis de Secuencia de ADN , Proteína Sequestosoma-1/químicaRESUMEN
In patients with postmenopausal osteoporosis low bone volume is associated with high bone marrow adipose tissue (MAT). Moreover, high MAT is associated with increased fracture risk. This suggests an interaction between MAT and bone turnover, however literature remains equivocal. Estrogen treatment decreases MAT, but the effect of raloxifene, a selective estrogen receptor modulator (SERM) registered for treatment of postmenopausal osteoporosis, on MAT is not known. The aim of this study is 1] to determine the effect of raloxifene on MAT and 2] to determine the relationship between MAT and bone turnover in patients with osteoporosis. Bone biopsies from the MORE trial were analyzed. The MORE trial investigated the effects of raloxifene 60 or 120mg per day versus placebo on bone metabolism and fracture incidence in patients with postmenopausal osteoporosis. We quantified MAT in iliac crest biopsies obtained at baseline and after 2years of treatment (n=53; age 68.2±6.2years). Raloxifene did not affect the change in MAT volume after 2years compared to baseline (placebo: 1.89±10.84%, raloxifene 60mg: 6.31±7.22%, raloxifene 120mg: -0.77±10.72%), nor affected change in mean adipocyte size (placebo: 1.45 (4.45) µm, raloxifene 60mg: 1.45 (4.35) µm, raloxifene 120mg: 0.81 (5.21) µm). Adipocyte number tended to decrease after placebo treatment (-9.92 (42.88) cells/mm2) and tended to increase during raloxifene 60mg treatment (13.27 (66.14) cells/mm2) while adipocyte number remained unchanged in the raloxifene 120mg group, compared to placebo (3.06 (39.80) cells/mm2, Kruskal-Wallis p=0.055, post hoc: placebo vs raloxifene 60mg p=0.017). MAT volume and adipocyte size were negatively associated with osteoclast number at baseline (R2=0.123, p=0.006 and R2=0.098, p=0.016 respectively). Furthermore adipocyte size was negatively associated with osteoid surface (R2=0.067, p=0.049). Finally, patients with vertebral fractures had higher MAT volume (50.82 (8.80)%) and larger adipocytes (55.75 (3.14) µm) compared to patients without fractures (45.58 (12.72)% p=0.032, 52.77 (3.73) µm p=0.004 respectively). In conclusion, raloxifene did not affect marrow adipose tissue, but tended to increase adipocyte number compared to placebo. At baseline MAT volume and adipocyte size were associated with bone resorption, and adipocyte size was associated with osteoid surface, suggesting an interaction between bone marrow adipocytes and bone turnover. In addition, we found that high MAT volume and larger adipocyte size are associated with prevalent vertebral fractures in postmenopausal women with osteoporosis, indicating that adipocyte size affects bone quality independent of bone volume.
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Tejido Adiposo/patología , Médula Ósea/patología , Remodelación Ósea/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Clorhidrato de Raloxifeno/uso terapéutico , Tejido Adiposo/efectos de los fármacos , Adiposidad/efectos de los fármacos , Anciano , Médula Ósea/efectos de los fármacos , Femenino , Humanos , Osteoporosis Posmenopáusica/complicaciones , Clorhidrato de Raloxifeno/farmacología , Fracturas de la Columna Vertebral/complicaciones , Fracturas de la Columna Vertebral/patologíaRESUMEN
Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2019;8:22-34.
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Células Madre Mesenquimatosas/citología , Osteopetrosis/metabolismo , Osteopetrosis/terapia , Ligando RANK/metabolismo , Biomimética/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Humanos , Células Madre Mesenquimatosas/fisiología , Osteopetrosis/genética , Ligando RANK/genética , Ingeniería de Tejidos/métodosRESUMEN
OBJECTIVE: Cartilage destruction in osteoarthritis (OA) is mediated mainly by matrix metalloproteinases (MMPs) and ADAMTS. The therapeutic candidature of targeting aggrecanases has not yet been defined in joints in which spontaneous OA arises from genetic susceptibility, as in the case of the STR/Ort mouse, without a traumatic or load-induced etiology. In addition, we do not know the long-term effect of aggrecanase inhibition on bone. We undertook this study to assess the potential aggrecanase selectivity of a variant of tissue inhibitor of metalloproteinases 3 (TIMP-3), called [-1A]TIMP-3, on spontaneous OA development and bone formation in STR/Ort mice. METHODS: Using the background of STR/Ort mice, which develop spontaneous OA, we generated transgenic mice that overexpress [-1A]TIMP-3, either ubiquitously or conditionally in chondrocytes. [-1A]TIMP-3 has an extra alanine at the N-terminus that selectively inhibits ADAMTS but not MMPs. We analyzed a range of OA-related measures in all mice at age 40 weeks. RESULTS: Mice expressing high levels of [-1A]TIMP-3 were protected against development of OA, while those expressing low levels were not. Interestingly, we also found that high levels of [-1A]TIMP-3 transgene overexpression resulted in increased bone mass, particularly in females. This regulation of bone mass was at least partly direct, as adult mouse primary osteoblasts infected with [-1A]TIMP-3 in vitro showed elevated rates of mineralization. CONCLUSION: The results provide evidence that [-1A]TIMP-3-mediated inhibition of aggrecanases can protect against cartilage degradation in a naturally occurring mouse model of OA, and they highlight a novel role that aggrecanase inhibition may play in increased bone mass.
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Densidad Ósea/genética , Cartílago Articular/enzimología , Endopeptidasas/metabolismo , Osteoartritis/enzimología , Inhibidores de Proteasas/metabolismo , Animales , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Osteoartritis/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5 fl/fl (Erk5 fl/fl) mice by ~6-8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5 fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5 fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5 fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.
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Proteína Quinasa 7 Activada por Mitógenos/fisiología , Osteoclastos/metabolismo , Columna Vertebral/anomalías , Animales , Resorción Ósea/genética , Hueso Esponjoso/anomalías , Catepsina K/biosíntesis , Recuento de Células , Femenino , Eliminación de Gen , Proteínas de Homeodominio/metabolismo , Integrasas/genética , Activación de Linfocitos , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 7 Activada por Mitógenos/genética , Factores de Transcripción NFATC/biosíntesis , Osteogénesis/genética , Receptor Activador del Factor Nuclear kappa-B/biosíntesis , Factores de Transcripción/metabolismoRESUMEN
Micro-CT analysis has become the standard method for assessing bone volume and architecture in small animals. However, micro-CT does not allow the assessment of bone turnover parameters such as bone formation rate and osteoclast (OC) number and surface. For these crucial variables histomorphometric analysis is still an essential technique. Histomorphometry however, is time consuming and, especially in mouse bones, OCs can be difficult to detect. The main purpose of this study was to develop and validate a relatively easy and rapid method to measure static and dynamic bone histomorphometry parameters. Here we present the adaptation of established staining protocols and three novel open source image analysis packages: TrapHisto, OsteoidHisto and CalceinHisto that allow rapid, semi-automated analysis of histomorphometric bone resorption, osteoid, and calcein double labelling parameters respectively. These three programs are based on ImageJ, but use a relatively simple user interface that hides the underlying complexity of the image analysis.
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Resorción Ósea/metabolismo , Programas Informáticos , Animales , Remodelación Ósea/fisiología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Osteogénesis/fisiologíaRESUMEN
Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specific bone formation and abnormal bone mineral content. A recent study suggested a role for the complement membrane attack complex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attack complex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture. In vivo bone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cortical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed in female mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis, with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bone cells were studied in isolation, in vitro osteoclastogenesis was significantly increased in male CD59a-deficient mice, although osteoblast formation was not altered. Our data reveal, for the first time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation in male mice results in longer and wider bones, but with less density, which is likely a major contributing factor for their susceptibility to osteoarthritis. These findings increase our understanding of the role of complement regulation in degenerative arthritis.
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Envejecimiento/patología , Huesos/patología , Antígenos CD59/metabolismo , Proteínas del Sistema Complemento/metabolismo , Eliminación de Gen , Caracteres Sexuales , Animales , Desarrollo Óseo , Células de la Médula Ósea/patología , Remodelación Ósea , Femenino , Masculino , Ratones Endogámicos C57BL , Tamaño de los Órganos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , FenotipoRESUMEN
OBJECTIVE: Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis. METHODS: OA was induced in wild-type (WT) and PAR2-deficient (PAR2-/-) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2-/- mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain. RESULTS: Osteophytes formed within 7â days post-DMM in WT mice but osteosclerosis was only evident from 14â days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2-/- mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2-/- mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2-/- mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone. CONCLUSIONS: This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.
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Artritis Experimental/patología , Huesos/patología , Cartílago Articular/patología , Osteoartritis/patología , Receptor PAR-2/metabolismo , Animales , Artralgia/etiología , Artralgia/patología , Artritis Experimental/etiología , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Osteoartritis/etiología , Osteocitos/metabolismoRESUMEN
INTRODUCTION: Inflammatory joint diseases such as rheumatoid arthritis are associated with local bone erosions and systemic bone loss, mediated by increased osteoclastic activity. The receptor activator of nuclear factor (NF) κB ligand (RANKL) plays a key role in mediating inflammation-induced bone loss, whereas tumour necrosis factor (TNF) plays a central role in the inflammatory process. Here we tested whether a recently identified class of small molecule inhibitors of RANKL signalling (ABD compounds) also affect TNF signalling and whether these compounds inhibit inflammation in an animal model of rheumatoid arthritis. METHODS: The inhibitory effects of the ABD compounds on TNF-induced signalling were tested in mouse macrophage cultures by western blotting and in an NFκB luciferase-reporter cell line. The anti-inflammatory effects of the compounds were tested in the mouse collagen-induced arthritis model of rheumatoid arthritis. RESULTS: The ABD compounds ABD328 and ABD345 both inhibited TNF-induced activation of the NFκB pathway and the extracellular signal-regulated kinase (ERK) and Jun kinase (JNK) mitogen activated protein kinases (MAPKs). When tested in the mouse collagen-induced arthritis model of rheumatoid arthritis, the compounds suppressed inflammatory arthritis, inhibited joint destruction and prevented systemic bone loss. Furthermore, one of the compounds (ABD328) showed oral activity. CONCLUSIONS: Here we describe a novel class of small molecule compounds that inhibit both RANKL- and TNF-induced NFκB and MAPK signalling in osteoclasts and macrophages, and inflammation and bone destruction in a mouse model of rheumatoid arthritis. These novel compounds therefore represent a promising new class of treatments for inflammatory diseases, such as rheumatoid arthritis.
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Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Compuestos de Bifenilo/farmacología , Resorción Ósea/metabolismo , Hexanonas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Ligando RANK/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Necrosis Tumoral/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Resorción Ósea/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , FN-kappa B/inmunología , FN-kappa B/metabolismo , Ligando RANK/inmunología , Ligando RANK/metabolismo , Factores de Necrosis Tumoral/inmunologíaRESUMEN
Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.
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Genes Dominantes , Genes Letales , Glaucoma/genética , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/genética , Alelos , Animales , Tipificación del Cuerpo , Dimerización , Heterocigoto , Ratones , Ratones Transgénicos , Mutación MissenseRESUMEN
Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.