RESUMEN
Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
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Resorción Ósea/patología , Osteoclastos/patología , Ligando RANK/metabolismo , Animales , Apoptosis , Resorción Ósea/metabolismo , Fusión Celular , Células Cultivadas , Humanos , Macrófagos/citología , Ratones , Osteocondrodisplasias/tratamiento farmacológico , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Osteoclastos/metabolismo , Transducción de SeñalRESUMEN
The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.
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Densidad Ósea/genética , Regulación de la Expresión Génica/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporosis/genética , Animales , Femenino , Ontología de Genes , Pleiotropía Genética , Estudio de Asociación del Genoma Completo , Genotipo , Masculino , Ratones , Ratones Transgénicos , Mutación , Osteoblastos/patología , Osteoclastos/patología , Osteoporosis/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Mapas de Interacción de Proteínas , Caracteres Sexuales , TranscriptomaRESUMEN
NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell-osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ-Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.
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Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Osteogénesis/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Apoptosis , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Cocultivo , Fragmentación del ADN , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Técnicas de Cultivo de Órganos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Neoplasias de la Próstata/patología , Ratas , Sesquiterpenos/farmacología , Transducción de Señal , Microtomografía por Rayos XRESUMEN
The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.
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Células de la Médula Ósea/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Western Blotting , Células de la Médula Ósea/citología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cannabinoides/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Humanos , Células MCF-7 , Ratones Noqueados , Osteoblastos/citología , Osteoclastos/citología , Osteogénesis/efectos de los fármacos , Osteólisis , Hormona Paratiroidea/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ligando RANK/farmacología , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Thyroid hormone (T(3)) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. Thrb(PV/PV) mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T(3) receptor (TRß(PV)) that cannot bind T(3) and interferes with the actions of wild-type TR. Thrb(PV/PV) mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and Thrb(PV/PV) mice. Activation of the canonical Wnt signaling in Thrb(PV/PV) mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T(3) treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T(3) inhibits the Wnt pathway by facilitating proteasomal degradation of ß-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in Thrb(PV/PV) mice, however, results from a gain of function for TRß(PV) that stabilizes ß-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T(3) and Wnt signaling pathways in the regulation of skeletal development and bone formation.
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Mutación , Osteoblastos/metabolismo , Osteogénesis/fisiología , Hipófisis/metabolismo , Receptores beta de Hormona Tiroidea/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Ratones , Ratones Mutantes , Osteoblastos/citología , Estabilidad Proteica , Receptores beta de Hormona Tiroidea/genética , Triyodotironina/metabolismo , Triyodotironina/farmacología , beta Catenina/genéticaRESUMEN
Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease.
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Estudios de Asociación Genética , Predisposición Genética a la Enfermedad/genética , Osteoartritis/genética , Animales , Huesos/patología , Sistemas CRISPR-Cas , Cartílago/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Edición Génica , Hormona Liberadora de Gonadotropina/genética , Yoduro Peroxidasa , Ratones , Ratones Noqueados , Osteoartritis/patología , Osteoartritis/cirugía , Factores de Transcripción Paired Box/genética , Fenotipo , Yodotironina Deyodinasa Tipo IIRESUMEN
Osteocytes are master regulators of the skeleton. We mapped the transcriptome of osteocytes from different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cellular-network. We define an osteocyte transcriptome signature of 1239 genes that distinguishes osteocytes from other cells. 77% have no previously known role in the skeleton and are enriched for genes regulating neuronal network formation, suggesting this programme is important in osteocyte communication. We evaluated 19 skeletal parameters in 733 knockout mouse lines and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human orthologs that cause monogenic skeletal disorders (P = 2.4 × 10-22) and are associated with the polygenic diseases osteoporosis (P = 1.8 × 10-13) and osteoarthritis (P = 1.6 × 10-7). Thus, we reveal the molecular landscape that regulates osteocyte network formation and function and establish the importance of osteocytes in human skeletal disease.
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Enfermedades Óseas/genética , Homeostasis , Osteocitos/metabolismo , Transcriptoma , Factores de Edad , Animales , Enfermedades Óseas/metabolismo , Huesos/metabolismo , Biología Computacional , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Osteocitos/citología , Osteoporosis/genética , Análisis de Secuencia de ARN , Factores SexualesRESUMEN
CONTEXT: The X-linked immunoglobulin superfamily, member 1 (IGSF1), gene is highly expressed in the hypothalamus and in pituitary cells of the POU1F1 lineage. Human loss-of-function mutations in IGSF1 cause central hypothyroidism, hypoprolactinemia, and macroorchidism. Additionally, most affected adults exhibit higher than average IGF-1 levels and anecdotal reports describe acromegaloid features in older subjects. However, somatotrope function has not yet been formally evaluated in this condition. OBJECTIVE: We aimed to evaluate the role of IGSF1 in human and murine somatotrope function. PATIENTS, DESIGN, AND SETTING: We evaluated 21 adult males harboring hemizygous IGSF1 loss-of-function mutations for features of GH excess, in an academic clinical setting. MAIN OUTCOME MEASURES: We compared biochemical and tissue markers of GH excess in patients and controls, including 24-hour GH profile studies in 7 patients. Parallel studies were undertaken in male Igsf1-deficient mice and wild-type littermates. RESULTS: IGSF1-deficient adult male patients demonstrated acromegaloid facial features with increased head circumference as well as increased finger soft-tissue thickness. Median serum IGF-1 concentrations were elevated, and 24-hour GH profile studies confirmed 2- to 3-fold increased median basal, pulsatile, and total GH secretion. Male Igsf1-deficient mice also demonstrated features of GH excess with increased lean mass, organ size, and skeletal dimensions and elevated mean circulating IGF-1 and pituitary GH levels. CONCLUSIONS: We demonstrate somatotrope neurosecretory hyperfunction in IGSF1-deficient humans and mice. These observations define a hitherto uncharacterized role for IGSF1 in somatotropes and indicate that patients with IGSF1 mutations should be evaluated for long-term consequences of increased GH exposure.
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Inmunoglobulinas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas de la Membrana/fisiología , Neurosecreción/fisiología , Somatotrofos/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Hormona del Crecimiento/biosíntesis , Humanos , Inmunoglobulinas/deficiencia , Factor I del Crecimiento Similar a la Insulina/análisis , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Masculino , Proteínas de la Membrana/deficiencia , Ratones , Persona de Mediana EdadRESUMEN
This chapter describes the use of point projection digital microradiography for rapid imaging and quantitation of bone mineral content in mice.
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Densidad Ósea , Huesos/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Microrradiografía/métodos , Animales , Huesos/fisiología , Procesamiento de Imagen Asistido por Computador/instrumentación , Ratones , Microrradiografía/instrumentación , Modelos Animales , Programas InformáticosRESUMEN
Background: Thyroid hormones act in bone and cartilage via thyroid hormone receptor alpha (TRα). In the absence of triiodothyronine (T3), TRα interacts with co-repressors, including nuclear receptor co-repressor-1 (NCoR1), which recruit histone deacetylases (HDACs) and mediate transcriptional repression. Dominant-negative mutations of TRα cause resistance to thyroid hormone alpha (RTHα; OMIM 614450), characterized by excessive repression of T3 target genes leading to delayed skeletal development, growth retardation, and bone dysplasia. Treatment with thyroxine has been of limited benefit, even in mildly affected individuals, and there is a need for new therapeutic strategies. It was hypothesized that (i) the skeletal manifestations of RTHα are mediated by the persistent TRα/NCoR1/HDAC repressor complex containing mutant TRα, and (ii) treatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) would ameliorate these manifestations. Methods: The skeletal phenotypes of (i) Thra1PV/+ mice, a well characterized model of RTHα; (ii) Ncor1ΔID/ΔID mice, which express an NCoR1 mutant that fails to interact with TRα; and (iii) Thra1PV/+Ncor1ΔID/ΔID double-mutant adult mice were determined. Wild-type, Thra1PV/+, Ncor1ΔID/ΔID, and Thra1PV/+Ncor1ΔID/ΔID double-mutant mice were also treated with SAHA to determine whether HDAC inhibition results in amelioration of skeletal abnormalities. Results:Thra1PV/+ mice had a severe skeletal dysplasia, characterized by short stature, abnormal bone morphology, and increased bone mineral content. Despite normal bone length, Ncor1ΔID/ΔID mice displayed increased cortical bone mass, mineralization, and strength. Thra1PV/+Ncor1ΔID/ΔID double-mutant mice displayed only a small improvement of skeletal abnormalities compared to Thra1PV/+ mice. Treatment with SAHA to inhibit histone deacetylation had no beneficial or detrimental effects on bone structure, mineralization, or strength in wild-type or mutant mice. Conclusions: These studies indicate treatment with SAHA is unlikely to improve the skeletal manifestations of RTHα. Nevertheless, the findings (i) confirm that TRα1 has a critical role in the regulation of skeletal development and adult bone mass, (ii) suggest a physiological role for alternative co-repressors that interact with TR in skeletal cells, and (iii) demonstrate a novel role for NCoR1 in the regulation of adult bone mass and strength.
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Co-Represor 1 de Receptor Nuclear/fisiología , Receptores alfa de Hormona Tiroidea/fisiología , Síndrome de Resistencia a Hormonas Tiroideas/genética , Animales , Densidad Ósea , Desarrollo Óseo , Calcificación Fisiológica , Ratones , Ratones Endogámicos C57BL , Síndrome de Resistencia a Hormonas Tiroideas/tratamiento farmacológico , Tiroxina/farmacología , Vorinostat/farmacologíaRESUMEN
OBJECTIVE: Bone loss in anorexia nervosa and following bariatric surgery is associated with an elevated circulating concentration of the gastrointestinal, anorexigenic hormone, peptide YY (PYY). Selective deletion of the PYY receptor Y1R in osteoblasts or Y2R in the hypothalamus results in high bone mass, but deletion of PYY in mice has resulted in conflicting skeletal phenotypes leading to uncertainty regarding its role in the regulation of bone mass. As PYY analogs are under development for treatment of obesity, we aimed to clarify the relationship between PYY and bone mass. METHODS: The skeletal phenotype of Pyy knockout (KO) mice was investigated during growth (postnatal day P14) and adulthood (P70 and P186) using X-ray microradiography, micro-CT, back-scattered electron scanning electron microscopy (BSE-SEM), histomorphometry and biomechanical testing. RESULTS: Bones from juvenile and Pyy KO mice were longer (Pâ¯<â¯0.001), with decreased bone mineral content (Pâ¯<â¯0.001). Whereas, bones from adult Pyy KO mice had increased bone mineral content (Pâ¯<â¯0.05) with increased mineralisation of both cortical (Pâ¯<â¯0.001) and trabecular (Pâ¯<â¯0.001) compartments. Long bones from adult Pyy KO mice were stronger (maximum load Pâ¯<â¯0.001), with increased stiffness (Pâ¯<â¯0.01) and toughness (Pâ¯<â¯0.05) compared to wild-type (WT) control mice despite increased cortical vascularity and porosity (Pâ¯<â¯0.001). The increased bone mass and strength in Pyy KO mice resulted from increases in trabecular (Pâ¯<â¯0.01) and cortical bone formation (Pâ¯<â¯0.05). CONCLUSIONS: These findings demonstrate that PYY acts as a negative regulator of osteoblastic bone formation, implicating increased PYY levels in the pathogenesis of bone loss during anorexia or following bariatric surgery.
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Huesos/anatomía & histología , Huesos/fisiología , Péptido YY/metabolismo , Animales , Densidad Ósea , Desarrollo Óseo , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Calcificación Fisiológica , Hueso Cortical/irrigación sanguínea , Hueso Cortical/ultraestructura , Femenino , Fémur/diagnóstico por imagen , Fémur/ultraestructura , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Osteoclastos/patología , PorosidadRESUMEN
Osteoporosis is characterized by low bone mineral density (BMD) and fragility fracture and affects over 200 million people worldwide. Bone quality describes the material properties that contribute to strength independently of BMD, and its quantitative analysis is a major priority in osteoporosis research. Tissue mineralization is a fundamental process requiring calcium and phosphate transporters. Here we identify impaired bone quality and strength in Slc20a2-/- mice lacking the phosphate transporter SLC20A2. Juveniles had abnormal endochondral and intramembranous ossification, decreased mineral accrual, and short stature. Adults exhibited only small reductions in bone mass and mineralization but a profound impairment of bone strength. Bone quality was severely impaired in Slc20a2-/- mice: yield load (-2.3 SD), maximum load (-1.7 SD), and stiffness (-2.7 SD) were all below values predicted from their bone mineral content as determined in a cohort of 320 wild-type controls. These studies identify Slc20a2 as a physiological regulator of tissue mineralization and highlight its critical role in the determination of bone quality and strength. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
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Huesos/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Animales , Animales Recién Nacidos , Desarrollo Óseo , Resorción Ósea/fisiopatología , Huesos/diagnóstico por imagen , Calcificación Fisiológica , Calcinosis/diagnóstico por imagen , Calcinosis/genética , Células Cultivadas , Condrocitos/metabolismo , Humanos , Incisivo/ultraestructura , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/metabolismo , Fenotipo , Cráneo/diagnóstico por imagen , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/deficiencia , Diente/crecimiento & desarrollo , Microtomografía por Rayos XRESUMEN
In the version of this article initially published, in Fig. 5a, the data in the right column of 'DAAM2 gRNA1' were incorrectly plotted as circles indicating 'untreated' rather than as squares indicating 'treated'. The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). We assessed genetic determinants of BMD as estimated by heel quantitative ultrasound in 426,824 individuals, identifying 518 genome-wide significant loci (301 novel), explaining 20% of its variance. We identified 13 bone fracture loci, all associated with estimated BMD (eBMD), in ~1.2 million individuals. We then identified target genes enriched for genes known to influence bone density and strength (maximum odds ratio (OR) = 58, P = 1 × 10-75) from cell-specific features, including chromatin conformation and accessible chromatin sites. We next performed rapid-throughput skeletal phenotyping of 126 knockout mice with disruptions in predicted target genes and found an increased abnormal skeletal phenotype frequency compared to 526 unselected lines (P < 0.0001). In-depth analysis of one gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This genetic atlas provides evidence linking associated SNPs to causal genes, offers new insight into osteoporosis pathophysiology, and highlights opportunities for drug development.
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Densidad Ósea/genética , Predisposición Genética a la Enfermedad/genética , Osteoporosis/genética , Adulto , Anciano , Animales , Femenino , Fracturas Óseas/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
NFκB is implicated in breast cancer bone metastasis and skeletal remodelling. However, the role of IKKß, a key component of the canonical NFκB pathway, in the regulation of breast cancer osteolytic metastasis has not been investigated. Here, we describe the cancer-specific contribution of IKKß to bone metastasis, skeletal tumour growth and osteolysis associated with breast cancer. IKKß is highly expressed in invasive breast tumours and its level of expression was higher in patients with bone metastasis. IKKß overexpression in parental MDA-MD-231 breast cancer cells, promoted mammary tumour growth but failed to convey osteolytic potential to these cells in mice. In contrast, IKKß overexpression in osteotropic sub-clones of MDA-MB-231 cells with differing osteolytic phenotypes increased incidence of bone metastasis, exacerbated osteolysis and enhanced skeletal tumour growth, whereas its knockdown was inhibitory. Functional and mechanistic studies revealed that IKKß enhanced the ability of osteotropic MDA-MB-231 cells to migrate, increase osteoclastogenesis, and to inhibit osteoblast differentiation via a mechanism mediated, at least in part, by cytoplasmic sequestering of FoxO3a and VEGFA production. Thus, tumour-selective manipulation of IKKß and its interaction with FoxO3a may represent a novel strategy to reduce the development of secondary breast cancer in the skeleton.
RESUMEN
The NFκB signaling pathway is implicated in breast cancer and bone metastasis. However, the bone-autonomous contribution of NFκB to breast cancer-induced osteolysis is poorly understood. Here, we report that pretreatment of osteoblasts with the sesquiterpene lactone Parthenolide (PTN), a verified NFκB inhibitor, prior to exposure to conditioned medium from human and mouse breast cancer cell lines enhanced osteoblast differentiation and reduced osteoblast ability to stimulate osteoclastogenesis. PTN prevented breast cancer-induced osteoclast formation and reduced the ability of breast cancer cells to prolong osteoclast survival and to inhibit osteoclast apoptosis. In vivo, administration of PTN in immuno-competent mice reduced osteolytic bone loss and skeletal tumour growth following injection of the syngeneic 4T1-BT1 cells and reduced local osteolysis caused by conditioned medium from human and mouse osteotropic breast cancer cell lines. Mechanistic studies revealed that NFκB inhibition by PTN in osteoblasts and osteoclasts was accompanied by a significant increase in ß-catenin activation and expression. Collectively, these results raise the possibility that combined targeting of NFκB and ß-catenin signalling in the tumour microenvironment may be of value in the treatment of breast cancer related osteolysis.
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Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , FN-kappa B/antagonistas & inhibidores , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteólisis/tratamiento farmacológico , Sesquiterpenos/farmacología , beta Catenina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteólisis/metabolismo , Osteólisis/patología , Transducción de Señal , Factores de TiempoRESUMEN
Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genome-wide association study (GWAS) in 142,487 individuals from the UK Biobank to identify loci associated with BMD as estimated by quantitative ultrasound of the heel. We identified 307 conditionally independent single-nucleotide polymorphisms (SNPs) that attained genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 previously unreported loci, and several rare variants with large effect sizes. To investigate the underlying mechanisms, we undertook (1) bioinformatic, functional genomic annotation and human osteoblast expression studies; (2) gene-function prediction; (3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and (4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. The results implicate GPC6 as a novel determinant of BMD, and also identify abnormal skeletal phenotypes in knockout mice associated with a further 100 prioritized genes.
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Densidad Ósea/genética , Calcáneo/patología , Estudio de Asociación del Genoma Completo , Osteoporosis/genética , Polimorfismo de Nucleótido Simple , Animales , Modelos Animales de Enfermedad , Femenino , Fémur/química , Perfilación de la Expresión Génica , Glipicanos/deficiencia , Glipicanos/genética , Glipicanos/fisiología , Trastornos del Crecimiento/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Anotación de Secuencia Molecular , Osteoblastos/metabolismo , Osteocondrodisplasias/congénito , Osteocondrodisplasias/genética , Osteoclastos/metabolismo , Osteocitos/metabolismo , Osteoporosis/patología , FenotipoRESUMEN
The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.