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The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains a public health concern and a subject of active research effort. Development of pre-clinical animal models is critical to study viral-host interaction, tissue tropism, disease mechanisms, therapeutic approaches, and long-term sequelae of infection. Here, we report two mouse models for studying SARS-CoV-2: A knock-in mAce2F83Y,H353K mouse that expresses a mouse-human hybrid form of the angiotensin-converting enzyme 2 (ACE2) receptor under the endogenous mouse Ace2 promoter, and a Rosa26 conditional knock-in mouse carrying the human ACE2 allele (Rosa26hACE2). Although the mAce2F83Y,H353K mice were susceptible to intranasal inoculation with SARS-CoV-2, they did not show gross phenotypic abnormalities. Next, we generated a Rosa26hACE2;CMV-Cre mouse line that ubiquitously expresses the human ACE2 receptor. By day 3 post infection with SARS-CoV-2, Rosa26hACE2;CMV-Cre mice showed significant weight loss, a variable degree of alveolar wall thickening and reduced survival rates. Viral load measurements confirmed inoculation in lung and brain tissues of infected Rosa26hACE2;CMV-Cre mice. The phenotypic spectrum displayed by our different mouse models translates to the broad range of clinical symptoms seen in the human patients and can serve as a resource for the community to model and explore both treatment strategies and long-term consequences of SARS-CoV-2 infection.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , SARS-CoV-2 , Animales , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/genética , COVID-19/patología , COVID-19/virología , Ratones , Humanos , SARS-CoV-2/genética , Ratones Transgénicos , Pulmón/virología , Pulmón/patología , Pulmón/metabolismo , Técnicas de Sustitución del GenRESUMEN
The expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR-34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR-34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR-34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast-specific overexpression of miR-34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR-34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1, and HEY2) and in p53-mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2, and HDAC1). More interestingly, we found that the metastatic-free survival probability was increased among a patient cohort from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) OS, which has lower expression of direct targets of miR-34c that was identified in our transcriptome analysis, such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR-34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR-34c in OS. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Loss-of-function mutations in DDRGK1 have been shown to cause Shohat type spondyloepimetaphyseal dysplasia (SEMD). In zebrafish, loss of function of ddrgk1 leads to defects in early cartilage development. Ddrgk1-/- mice show delayed mesenchymal condensation in the limb buds and early embryonic lethality. Mechanistically, Ddrgk1 interacts with Sox9 and reduces ubiquitin-mediated proteasomal degradation of Sox9 protein. To investigate the cartilage-specific role of DDRGK1, conditional knockout mice were generated by intercrossing Prx1-Cre transgenic mice with Ddrgkfl/fl mice to delete its expression in limb mesenchymal cells. Mutant mice showed progressive severe shortening of the limbs and joint abnormalities. The growth plate showed disorganization with shortened proliferative zone and enlarged hypertrophic zone. In correlation with these findings, Sox9 and Col2a1 protein levels were decreased, while Col10a1 expression was expanded. These data demonstrate the importance of Ddrgk1 during growth plate development. In contrast, deletion of Ddrgk1 with the osteoblast-specific Osteocalcin-Cre and Leptin receptor-Cre lines did not show bone phenotypes, suggesting that the effect on limb development is cartilage-specific. To evaluate the role of DDRGK1 in cartilage postnatal homeostasis, inducible Agc1-CreERT2; Ddrgklfl/fl mice were generated. Mice in which Ddrgk1 was deleted at 3 months of age showed disorganized growth plate, with significant reduction in proteoglycan deposition. These data demonstrate a postnatal requirement for Ddrgk1 in maintaining normal growth plate morphology. Together, these findings highlight the physiological role of Ddrgk1 in the development and maintenance of the growth plate cartilage. Furthermore, these genetic mouse models recapitulate the clinical phenotype of short stature and joint abnormalities observed in patients with Shohat type SEMD.
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Placa de Crecimiento , Pez Cebra , Animales , Cartílago , Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis , Placa de Crecimiento/metabolismo , Ratones , Ratones Transgénicos , OsteocondrodisplasiasRESUMEN
Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.
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Difosfonatos , Nitrógeno , Alendronato/farmacología , Animales , Huesos , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Humanos , Ratones , OsteoclastosRESUMEN
Skeletal development is a complex process which requires the tight regulation of gene activation and suppression in response to local signaling pathways. Among these pathways, Notch signaling is implicated in governing cell fate determination, proliferation, differentiation and apoptosis of skeletal cells-osteoblasts, osteoclasts, osteocytes and chondrocytes. Moreover, human genetic mutations in Notch components emphasize the critical roles of Notch signaling in skeletal development and homeostasis. In this review, we focus on the physiological roles of Notch signaling in skeletogenesis, postnatal bone and cartilage homeostasis and fracture repair. We also discuss the pathological gain- and loss-of-function of Notch signaling in bone and cartilage, resulting in osteosarcoma and age-related degenerative diseases, such as osteoporosis and osteoarthritis. Understanding the physiological and pathological function of Notch signaling in skeletal tissues using animal models and human genetics will provide new insights into disease pathogenesis and offer novel approaches for the treatment of bone/cartilage diseases.
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Enfermedades Óseas/metabolismo , Condrogénesis , Osteogénesis , Receptores Notch/metabolismo , Transducción de Señal , Animales , Enfermedades Óseas/patología , Enfermedades Óseas/fisiopatología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/fisiopatología , Homeostasis , Humanos , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Bone metastasis is a major health threat to breast cancer patients. Tumor-derived Jagged1 represents a central node in mediating tumor-stromal interactions that promote osteolytic bone metastasis. Here, we report the development of a highly effective fully human monoclonal antibody against Jagged1 (clone 15D11). In addition to its inhibitory effect on bone metastasis of Jagged1-expressing tumor cells, 15D11 dramatically sensitizes bone metastasis to chemotherapy, which induces Jagged1 expression in osteoblasts to provide a survival niche for cancer cells. We further confirm the bone metastasis-promoting function of osteoblast-derived Jagged1 using osteoblast-specific Jagged1 transgenic mouse model. These findings establish 15D11 as a potential therapeutic agent for the prevention or treatment of bone metastasis.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Proteína Jagged-1/antagonistas & inhibidores , Animales , Humanos , Ratones , Ratones Transgénicos , Osteoblastos/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pathogenic variants in genes encoding components of the BRG1-associated factor (BAF) chromatin remodeling complex have been associated with intellectual disability syndromes. We identified heterozygous, novel variants in ACTL6A, a gene encoding a component of the BAF complex, in three subjects with varying degrees of intellectual disability. Two subjects have missense variants affecting highly conserved amino acid residues within the actin-like domain. Missense mutations in the homologous region in yeast actin were previously reported to be dominant lethal and were associated with impaired binding of the human ACTL6A to ß-actin and BRG1. A third subject has a splicing variant that creates an in-frame deletion. Our findings suggest that the variants identified in our subjects may have a deleterious effect on the function of the protein by disturbing the integrity of the BAF complex. Thus, ACTL6A gene mutation analysis should be considered in patients with intellectual disability, learning disabilities, or developmental language disorder.
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Actinas/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/genética , Mutación Missense/genética , Adolescente , Niño , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Exoma , Cara , Femenino , Deformidades Congénitas de la Mano/fisiopatología , Heterocigoto , Humanos , Discapacidad Intelectual/fisiopatología , Masculino , Micrognatismo/genética , Micrognatismo/fisiopatología , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Osteocytes are the terminally differentiated cell type of the osteoblastic lineage and have important functions in skeletal homeostasis. Although the transcriptional regulation of osteoblast differentiation has been well characterized, the factors that regulate differentiation of osteocytes from mature osteoblasts are poorly understood. Here we show that miR-23aâ¼27aâ¼24-2 (miR-23a cluster) promotes osteocyte differentiation. Osteoblast-specific miR-23a cluster gain-of-function mice have low bone mass associated with decreased osteoblast but increased osteocyte numbers. By contrast, loss-of-function transgenic mice overexpressing microRNA decoys for either miR-23a or miR-27a, but not miR24-2, show decreased osteocyte numbers. Moreover, RNA-sequencing analysis shows altered transforming growth factor-ß (TGF-ß) signalling. Prdm16, a negative regulator of the TGF-ß pathway, is directly repressed by miR-27a with concomitant alteration of sclerostin expression, and pharmacological inhibition of TGF-ß rescues the phenotypes observed in the gain-of-function transgenic mice. Taken together, the miR-23a cluster regulates osteocyte differentiation by modulating the TGF-ß signalling pathway through targeting of Prdm16.
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Diferenciación Celular/genética , Regulación de la Expresión Génica , MicroARNs/genética , Osteocitos/metabolismo , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/genética , Animales , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Femenino , Células HEK293 , Humanos , Ratones Transgénicos , Modelos Genéticos , Familia de Multigenes , Osteocitos/citología , Osteogénesis/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1-/- mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.
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Proteínas Portadoras/fisiología , Osteocondrodisplasias/genética , Factor de Transcripción SOX9/metabolismo , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales , Animales , Cartílago/crecimiento & desarrollo , Células Cultivadas , Condrogénesis , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Ratones Noqueados , Mutación , Osteocondrodisplasias/metabolismo , Linaje , Isoformas de Proteínas/fisiología , Estabilidad Proteica , Sitios de Empalme de ARN , Pez CebraRESUMEN
Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.
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Neoplasias Óseas , Arteria Ilíaca , Metástasis de la Neoplasia , Animales , Huesos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Miembro Posterior , HumanosRESUMEN
Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development.
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Densidad Ósea/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Huesos/efectos de los fármacos , Condrocitos/efectos de los fármacos , Losartán/farmacología , Angiotensinas/efectos de los fármacos , Angiotensinas/metabolismo , Animales , Densidad Ósea/fisiología , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Cartílago/efectos de los fármacos , Diferenciación Celular , Condrocitos/fisiología , Femenino , Placa de Crecimiento/efectos de los fármacos , Hipertrofia/etiología , Losartán/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Fosforilación , Ligando RANK/antagonistas & inhibidores , Ligando RANK/metabolismo , Células RAW 264.7 , RadiografíaRESUMEN
RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.
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Desarrollo Óseo , Mutación , RecQ Helicasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Canal Anal/anomalías , Canal Anal/metabolismo , Canal Anal/patología , Animales , Craneosinostosis/genética , Craneosinostosis/metabolismo , Craneosinostosis/patología , Enanismo/genética , Enanismo/metabolismo , Enanismo/patología , Defectos del Tabique Interatrial/genética , Defectos del Tabique Interatrial/metabolismo , Defectos del Tabique Interatrial/patología , Humanos , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/metabolismo , Deformidades Congénitas de las Extremidades/patología , Ratones , Ratones Transgénicos , Rótula/anomalías , Rótula/metabolismo , Rótula/patología , Radio (Anatomía)/anomalías , Radio (Anatomía)/metabolismo , Radio (Anatomía)/patología , RecQ Helicasas/genética , Síndrome Rothmund-Thomson/genética , Síndrome Rothmund-Thomson/metabolismo , Síndrome Rothmund-Thomson/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The skeleton originates from stem cells residing in the sclerotome and neural crest that undergo proliferation, migration, and commitment. The development of the skeletal stem cells is influenced by many signaling pathways that govern cell fate determination, proliferation, differentiation, and apoptosis. This review will focus on Notch signaling functions in regulating the different cell types that form the skeletal system as well as the interplay between them to maintain homeostasis. Osteochondroprogenitors require Notch signaling to maintain multipotency and to prevent premature differentiation into osteoblasts. Subsequently, overactivation of Notch signaling suppresses osteoblast maturation. Moreover, Notch signaling in osteochondroprogenitors is required for chondrocyte proliferation and hypertrophy and suppresses terminal differentiation. Translational studies demonstrated a crucial role of Notch signaling in osteosarcoma and osteoarthritis, where concepts derived from developmental pathways are often recapitulated. This brings hope of taking advantage of the molecular mechanisms learned from development to approach the pathological processes underlying abnormal bone/cartilage metabolism or tumorigenesis. Pharmacological agents that target Notch receptors or ligands in a tissue-specific fashion would offer new opportunities for treating bone/cartilage diseases caused by dysregulation of Notch signaling.
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Músculo Esquelético/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Células Madre/citología , Animales , Huesos/citología , Huesos/metabolismo , Diferenciación Celular/fisiología , Humanos , Células Madre/metabolismoRESUMEN
TGF-ß is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-ß bioavailability by attenuating maturation of pro-TGF-ß during cartilage homeostasis. However, whether regulation of intracellular TGF-ß maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-ß/pro-TGF-ß ratio, with increased expression of TGF-ß downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-ß antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-ß signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-ß maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.
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Remodelación Ósea , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Sialoglicoproteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Anticuerpos/farmacología , Enfermedades Óseas Metabólicas/complicaciones , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Enfermedades Óseas Metabólicas/fisiopatología , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/genética , Resorción Ósea/complicaciones , Resorción Ósea/genética , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Calcificación Fisiológica/efectos de los fármacos , Calcificación Fisiológica/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Células Cultivadas , Fémur/diagnóstico por imagen , Fémur/efectos de los fármacos , Fémur/patología , Fémur/fisiopatología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Homeostasis/efectos de los fármacos , Ratones , Tamaño de los Órganos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fenotipo , Radiografía , Receptores de Factores de Crecimiento de Fibroblastos/deficiencia , Sialoglicoproteínas/deficiencia , Transducción de Señal/genéticaRESUMEN
Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P(2) levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P(2) signaling in skeletal development and maintenance.
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Desarrollo Óseo/genética , Displasia Cleidocraneal/genética , Displasia Ectodérmica/genética , Flavoproteínas/genética , Predisposición Genética a la Enfermedad/genética , Deformidades Congénitas de las Extremidades/genética , Micrognatismo/genética , Animales , Secuencia de Bases , Displasia Cleidocraneal/patología , Displasia Ectodérmica/patología , Exoma/genética , Fibroblastos , Mutación del Sistema de Lectura/genética , Genotipo , Humanos , Deformidades Congénitas de las Extremidades/patología , Ratones , Micrognatismo/patología , Datos de Secuencia Molecular , Mutación Missense/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas , Análisis de Secuencia de ADNRESUMEN
Notch signaling plays a critical role during development by directing the binary cell fate decision between progenitors and differentiated cells. Previous studies have shown sustained Notch activation in cartilage leads to chondrodysplasia. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. However, it is still unknown how Notch governs chondrogenesis in the axial skeleton where Notch serves a primary patterning function. We hypothesized that both Rbpj-dependent and Rbpj-independent Notch signaling mechanisms might be involved. Cartilage-specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by "butterfly" vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj-deficient Notch GOF mice compared with Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. To further understand the molecular basis of this regulation, we performed chromatin immunoprecipitation (ChIP) assays and detected the recruitment of the Rbpj/NICD transcription complex to Rbpj-binding sites upstream of the Sox9 promoter. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Hence, Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis.
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Diferenciación Celular/fisiología , Condrocitos/citología , Regulación de la Expresión Génica/fisiología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Receptores Notch/fisiología , Factor de Transcripción SOX9/genética , Animales , Inmunoprecipitación de Cromatina , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Dysosteosclerosis (DSS) is the form of osteopetrosis distinguished by the presence of skin findings such as red-violet macular atrophy, platyspondyly and metaphyseal osteosclerosis with relative radiolucency of widened diaphyses. At the histopathological level, there is a paucity of osteoclasts when the disease presents. In two patients with DSS, we identified homozygous or compound heterozygous missense mutations in SLC29A3 by whole-exome sequencing. This gene encodes a nucleoside transporter, mutations in which cause histiocytosis-lymphadenopathy plus syndrome, a group of conditions with little or no skeletal involvement. This transporter is essential for lysosomal function in mice. We demonstrate the expression of Slc29a3 in mouse osteoclasts in vivo. In monocytes from patients with DSS, we observed reduced osteoclast differentiation and function (demineralization of calcium surface). Our report highlights the pleomorphic consequences of dysfunction of this nucleoside transporter, and importantly suggests a new mechanism for the control of osteoclast differentiation and function.
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Exoma , Mutación , Proteínas de Transporte de Nucleósidos/genética , Osteopetrosis/genética , Osteosclerosis/genética , Secuencia de Aminoácidos , Animales , Preescolar , Consanguinidad , Femenino , Humanos , Lactante , Ratones , Datos de Secuencia Molecular , Osteopetrosis/diagnóstico por imagen , Osteosclerosis/diagnóstico por imagen , Radiografía , Alineación de SecuenciaRESUMEN
During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.
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Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Osteoblastos/fisiología , Osteogénesis , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transducción de Señal , Animales , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Proteína Jagged-1 , Ratones , Ratones Transgénicos , MicroARNs/genética , Osteoblastos/citología , Osteoclastos/fisiología , Osteoporosis/metabolismo , Osteoporosis/patología , Osteosarcoma/patología , Proteínas Serrate-JaggedRESUMEN
The phenobarbital (PB) responsive enhancers in CYP2B genes contain a core of two direct repeat-4 nuclear receptor binding sites, NR-1 and NR-2, which flank an NF-1 site and appear to be most important for PB responsiveness. Additional sequences outside the core are required for maximal PB responsiveness, including a third direct repeat-4 site, NR-3. The PB response is mediated by constitutive androstane receptor (CAR) which binds as a CAR/RXR heterodimer to the NR sites. To determine the relative importance of the third NR site, each of the NR sites was mutated individually and in all combinations in the rat PB responsive unit (PBRU). Mutation of NR-3 resulted in similar effects on transactivation of the PBRU by CAR in HepG2 cells as did mutations of NR-1 and NR-2. The recruitment of GRIP1/SRC-2 by CAR/RXR to the PBRU assessed by gel shift assays was cooperatively enhanced if more than one NR site in the PBRU was occupied by CAR/RXR. NR-3 in combination with NR-1 or NR-2 was equal to NR-1 and NR-2 in mediating this cooperative recruitment. Recruitment of SRC-1 and GRIP1/SRC-2 was similar for all NR sites, while some selectivity of NR-1 for SRC-3 was observed. SRC-3 also exhibited CAR-independent activation of the PBRU in HepG2 cells. Micrococcal nuclease mapping of nucleosomes revealed that the NR-1/NR-2 core of the PBRU is present in a nucleosome while NR-3 is present in the linker adjacent to the nucleosome. In the linear sequence NR-3 is further from NR-1 than NR-2 is, but in a nucleosomal structure, NR-3 is well positioned for cooperative recruitment of GRIP1/SRC-2 by CAR/RXR that is bound to NR-3 and either NR-1 or NR-2, while NR-1 and NR-2 are on opposite sides of the nucleosome separated by the histone core. These results demonstrate that NR-3 is functionally similar to NR-1 and NR-2 in CAR transactivation of the PBRU in vitro and suggest that NR-3 may have a greater role in a chromatin context in vivo than is apparent from transient transfection studies.
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Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores X Retinoide/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Receptor de Androstano Constitutivo , Dimerización , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Nucleosomas/química , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Receptores X Retinoide/química , Receptores X Retinoide/genética , Análisis de Secuencia de ADN , Factores de Transcripción/química , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Post-translational modification of chromatin has profound effects on many biological processes including transcriptional regulation, heterochromatin organization, and X-chromosome inactivation. Recent studies indicate that methylation on specific histone lysine (K) residues participates in many of these processes. Lysine methylation occurs in three distinct states, having either one (me1), two (me2) or three (me3) methyl groups attached to the amine group of the lysine side chain. These differences in modification state have an important role in defining how methylated chromatin is recognized and interpreted. Until recently, histone lysine methylation was considered a stable modification, but the identification of histone demethylase enzymes has demonstrated the reversibility of this epigenetic mark. So far, all characterized histone demethylases show enzymatic activity towards lysine residues modified in the me1 or me2 state, leaving open the possibility that me3 constitutes an irreversible modification. Here we demonstrate that JHDM3A (jumonji C (JmjC)-domain-containing histone demethylase 3A; also known as JMJD2A) is capable of removing the me3 group from modified H3 lysine 9 (H3K9) and H3 lysine 36 (H3K36). Overexpression of JHDM3A abrogates recruitment of HP1 (heterochromatin protein 1) to heterochromatin, indicating a role for JHDM3A in antagonizing methylated H3K9 nucleated events. siRNA-mediated knockdown of JHDM3A leads to increased levels of H3K9 methylation and upregulation of a JHDM3A target gene, ASCL2, indicating that JHDM3A may function in euchromatin to remove histone methylation marks that are associated with active transcription.