RESUMEN
Thyroid hormones (TH) are essential for skeletal development and adult bone homeostasis. Their bioavailability is determined by specific transporter proteins at the cell surface. The TH-specific transporter monocarboxylate transporter 8 (MCT8) was recently reported as a regulator of bone mass in mice. Given that high systemic triiodothyronine (T3) levels in Mct8 knockout (KO) mice are still able to cause trabecular bone loss, alternative TH transporters must substitute for MCT8 function in bone. In this study, we analyzed the skeletal phenotypes of male Oatp1c1 KO and Mct10 KO mice, which are euthyroid, and male Mct8/Oatp1c1 and Mct8/Mct10 double KO mice, which have elevated circulating T3 levels, to unravel the role of TH transport in bone. MicroCT analysis showed no significant trabecular bone changes in Oatp1c1 KO mice at 4 weeks and 16 weeks of age compared with wild-type littermate controls, whereas 16-week-old Mct8/Oatp1c1 double KO animals displayed trabecular bone loss. At 12 weeks, Mct10 KO mice, but not Mct8/Mct10 double KO mice, had decreased trabecular femoral bone volume with reduced osteoblast numbers. By contrast, lack of Mct10 in 24-week-old mice led to trabecular bone gain at the femur with increased osteoblast numbers and decreased osteoclast numbers whereas Mct8/Mct10 double KO did not alter bone mass. Neither Mct10 nor Mct8/Mct10 deletion affected vertebral bone structures at both ages. In vitro, osteoblast differentiation and activity were impaired by Mct10 and Mct8/Mct10-deficiency. These data demonstrate that MCT10, but not OATP1C1, is a site- and age-dependent regulator of bone mass and turnover in male mice.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Huesos/metabolismo , Animales , Transporte Biológico , Fenómenos Biomecánicos , Hueso Esponjoso/metabolismo , Diferenciación Celular , Fémur/fisiología , Homeostasis , Masculino , Ratones , Ratones Noqueados , Proteínas de Transporte de Catión Orgánico/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteocitos/citología , Fenotipo , Simportadores/metabolismo , Glándula Tiroides/metabolismo , Hormonas Tiroideas/metabolismo , Triyodotironina/metabolismo , Microtomografía por Rayos XRESUMEN
A synoptic overview of scientific methods applied in bone and associated research fields across species has yet to be published. Experts from the EU Cost Action GEMSTONE ("GEnomics of MusculoSkeletal Traits translational Network") Working Group 2 present an overview of the routine techniques as well as clinical and research approaches employed to characterize bone phenotypes in humans and selected animal models (mice and zebrafish) of health and disease. The goal is consolidation of knowledge and a map for future research. This expert paper provides a comprehensive overview of state-of-the-art technologies to investigate bone properties in humans and animals - including their strengths and weaknesses. New research methodologies are outlined and future strategies are discussed to combine phenotypic with rapidly developing -omics data in order to advance musculoskeletal research and move towards "personalised medicine".
Asunto(s)
Huesos/metabolismo , Genómica/métodos , Fenómenos Fisiológicos Musculoesqueléticos/genética , Animales , Huesos/patología , Redes Reguladoras de Genes/fisiología , Humanos , Ratones , Modelos Animales , Fenotipo , Proteómica/métodos , Pez CebraRESUMEN
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
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.
Asunto(s)
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
The development of the craniofacial skeleton relies on complex temporospatial organization of diverse cell types by key signalling molecules. Even minor disruptions to these processes can result in deleterious consequences for the structure and function of the skull. Thyroid hormone deficiency causes delayed craniofacial and tooth development, dysplastic facial features and delayed development of the ossicles in the middle ear. Thyroid hormone excess, by contrast, accelerates development of the skull and, in severe cases, might lead to craniosynostosis with neurological sequelae and facial hypoplasia. The pathogenesis of these important abnormalities remains poorly understood and underinvestigated. The orchestration of craniofacial development and regulation of suture and synchondrosis growth is dependent on several critical signalling pathways. The underlying mechanisms by which these key pathways regulate craniofacial growth and maturation are largely unclear, but studies of single-gene disorders resulting in craniofacial malformations have identified a number of critical signalling molecules and receptors. The craniofacial consequences resulting from gain-of-function and loss-of-function mutations affecting insulin-like growth factor 1, fibroblast growth factor receptor and WNT signalling are similar to the effects of altered thyroid status and mutations affecting thyroid hormone action, suggesting that these critical pathways interact in the regulation of craniofacial development.
Asunto(s)
Anomalías Craneofaciales/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Craneosinostosis/metabolismo , Humanos , Transducción de Señal/fisiología , Cráneo/metabolismoRESUMEN
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.
Asunto(s)
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
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 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.
Asunto(s)
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
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.
Asunto(s)
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
Levothyroxine (LT4) is a form of thyroid hormone used to treat hypothyroidism. In the brain, T4 is converted to the active form T3 by type 2 deiodinase (D2). Thus, it is intriguing that carriers of the Thr92Ala polymorphism in the D2 gene (DIO2) exhibit clinical improvement when liothyronine (LT3) is added to LT4 therapy. Here, we report that D2 is a cargo protein in ER Golgi intermediary compartment (ERGIC) vesicles, recycling between ER and Golgi. The Thr92-to-Ala substitution (Ala92-D2) caused ER stress and activated the unfolded protein response (UPR). Ala92-D2 accumulated in the trans-Golgi and generated less T3, which was restored by eliminating ER stress with the chemical chaperone 4-phenyl butyric acid (4-PBA). An Ala92-Dio2 polymorphism-carrying mouse exhibited UPR and hypothyroidism in distinct brain areas. The mouse refrained from physical activity, slept more, and required additional time to memorize objects. Enhancing T3 signaling in the brain with LT3 improved cognition, whereas restoring proteostasis with 4-PBA eliminated the Ala92-Dio2 phenotype. In contrast, primary hypothyroidism intensified the Ala92-Dio2 phenotype, with only partial response to LT4 therapy. Disruption of cellular proteostasis and reduced Ala92-D2 activity may explain the failure of LT4 therapy in carriers of Thr92Ala-DIO2.
Asunto(s)
Encéfalo , Estrés del Retículo Endoplásmico , Hipotiroidismo , Yoduro Peroxidasa , Polimorfismo Genético , Respuesta de Proteína Desplegada , Sustitución de Aminoácidos , Animales , Encéfalo/enzimología , Encéfalo/patología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Aparato de Golgi/enzimología , Aparato de Golgi/genética , Células HEK293 , Humanos , Hipotiroidismo/tratamiento farmacológico , Hipotiroidismo/enzimología , Hipotiroidismo/genética , Hipotiroidismo/patología , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Ratones , Ratones Transgénicos , Mutación Missense , Tiroxina/uso terapéutico , Triyodotironina/uso terapéutico , Yodotironina Deyodinasa Tipo IIRESUMEN
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.
Asunto(s)
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
T3 is an important regulator of skeletal development and adult bone maintenance. Thyroid hormone action requires efficient transport of T4 and T3 into target cells. We hypothesized that monocarboxylate transporter (MCT) 8, encoded by Mct8 on the X-chromosome, is an essential thyroid hormone transporter in bone. To test this hypothesis, we determined the juvenile and adult skeletal phenotypes of male Mct8 knockout mice (Mct8KO) and Mct8D1D2KO compound mutants, which additionally lack the ability to convert the prohormone T4 to the active hormone T3. Prenatal skeletal development was normal in both Mct8KO and Mct8D1D2KO mice, whereas postnatal endochondral ossification and linear growth were delayed in both Mct8KO and Mct8D1D2KO mice. Furthermore, bone mass and mineralization were decreased in adult Mct8KO and Mct8D1D2KO mice, and compound mutants also had reduced bone strength. Delayed bone development and maturation in Mct8KO and Mct8D1D2KO mice is consistent with decreased thyroid hormone action in growth plate chondrocytes despite elevated serum T3 concentrations, whereas low bone mass and osteoporosis reflects increased thyroid hormone action in adult bone due to elevated systemic T3 levels. These studies identify an essential physiological requirement for MCT8 in chondrocytes, and demonstrate a role for additional transporters in other skeletal cells during adult bone maintenance.
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Huesos/metabolismo , Proteínas de Transporte de Membrana/fisiología , Animales , Desarrollo Óseo/genética , Huesos/embriología , Huesos/fisiología , Condrocitos/fisiología , Desarrollo Fetal/genética , Yoduro Peroxidasa/genética , Masculino , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transportadores de Ácidos Monocarboxílicos , Fenotipo , Simportadores , Enfermedades de la Tiroides/genética , Enfermedades de la Tiroides/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiología , Hormonas Tiroideas/sangre , Yodotironina Deyodinasa Tipo IIRESUMEN
Craniosynostosis is a developmental disorder of the skull arising from premature bony fusion of cranial sutures, the sites of skull bone growth. In a recent gene microarray study, we demonstrated that retinol-binding protein 4 (RBP4) was the most highly downregulated gene in suture tissue during the pathological process of premature bony fusion. To gain insight into the function of RBP4 in cranial sutures, we analysed primary cells cultured from human cranial suture mesenchyme. These cells express RBP4 but not CRBP1, cellular retinol-binding protein 1, the typical cytoplasmic retinol storage protein. Using flow cytometry, we showed that suture mesenchymal cells express the RBP4 receptor, STRA6, on the cell surface. In a cell culture model of cranial osteogenesis, we found that RBP4 was significantly downregulated during mineralization, analogous to its decrease in pathological suture fusion. We found that cranial suture cells do not secrete detectable levels of RBP4, suggesting that it acts in a cell-autonomous manner. High-resolution confocal microscopy with a panel of antibody markers of cytoplasmic organelles demonstrated that RBP4 was present in several hundred cytoplasmic vesicles of about 300 nm in diameter which, in large part, were conspicuously distinct from the ER, the Golgi and endosomes of the endocytic pathway. We speculate that in suture mesenchymal cells, endogenous RBP4 receives retinol from STRA6 and the RBP4-retinol complex is stored in vesicles until needed for conversion to retinoic acid in the process of osteogenesis. This study extends the role of RBP4 beyond that of a serum transporter of retinol and implicates a broader role in osteogenesis.
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Suturas Craneales/metabolismo , Craneosinostosis/metabolismo , Vesículas Citoplasmáticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Células Cultivadas , Suturas Craneales/patología , Craneosinostosis/genética , Craneosinostosis/patología , Vesículas Citoplasmáticas/patología , Regulación hacia Abajo , Citometría de Flujo , Humanos , Proteínas de la Membrana/metabolismo , Células Madre Mesenquimatosas/patología , Microscopía Confocal , Microscopía Fluorescente , Tamaño de los Orgánulos , Osteogénesis/genética , Cultivo Primario de Células , Proteínas Plasmáticas de Unión al Retinol/genética , Factores de Tiempo , Vitamina A/metabolismoRESUMEN
Leucocytes are essential in healing wounds and are predominantly involved in the inflammatory and granulation stages of wound repair. Eosinophils are granulocytic leucocytes and are specifically regulated by interleukin-5 (IL-5), a cytokine produced by T helper 2 (Th2) cells. To characterize more clearly the role of the IL-5 and eosinophils in the wound healing process, IL-5-overexpressing and IL-5-deficient mice were used as models of eosinophilia and eosinophil depletion, respectively. Our results reveal a significantly altered inflammatory response between IL-5-overexpressing and IL-5 knockout mice post-wounding. Healing was significantly delayed in IL-5-overexpressing mice with wounds gaping wider and exhibiting impaired re-epithelialization. A delay in collagen deposition was observed suggesting a direct effect on matrix synthesis. A significant increase in inflammatory cell infiltration, particularly eosinophils and CD4(+) cells, one of the main cell types which secrete IL-5, was observed in IL-5-overexpressing mice wounds suggesting that one of the main roles of IL-5 in wound repair may be to promote the infiltration of eosinophils into healing wounds. Healing is delayed in IL-5-overexpressing mice and this corresponds to significantly increased levels of eosinophils and CD4(+) cells within the wound site that may contribute to and exacerbate the inflammatory response, resulting in detrimental wound repair.