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1.
Cell ; 147(6): 1324-39, 2011 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-22153076

RESUMEN

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Querubismo/metabolismo , Transducción de Señal , Tanquirasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Querubismo/genética , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Estabilidad Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Eliminación de Secuencia , Quinasa Syk , Tanquirasas/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación
2.
J Med Genet ; 61(2): 117-124, 2024 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-37399313

RESUMEN

BACKGROUND: Otosclerosis is a common cause of adult-onset progressive hearing loss, affecting 0.3%-0.4% of the population. It results from dysregulation of bone homeostasis in the otic capsule, most commonly leading to fixation of the stapes bone, impairing sound conduction through the middle ear. Otosclerosis has a well-known genetic predisposition including familial cases with apparent autosomal dominant mode of inheritance. While linkage analysis and genome-wide association studies suggested an association with several genomic loci and with genes encoding structural proteins involved in bone formation or metabolism, the molecular genetic pathophysiology of human otosclerosis is yet mostly unknown. METHODS: Whole-exome sequencing, linkage analysis, generation of CRISPR mutant mice, hearing tests and micro-CT. RESULTS: Through genetic studies of kindred with seven individuals affected by apparent autosomal dominant otosclerosis, we identified a disease-causing variant in SMARCA4, encoding a key component of the PBAF chromatin remodelling complex. We generated CRISPR-Cas9 transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue. Mutant Smarca4+/E1548K mice exhibited marked hearing impairment demonstrated through acoustic startle response and auditory brainstem response tests. Isolated ossicles of the auditory bullae of mutant mice exhibited a highly irregular structure of the incus bone, and their in situ micro-CT studies demonstrated the anomalous structure of the incus bone, causing disruption in the ossicular chain. CONCLUSION: We demonstrate that otosclerosis can be caused by a variant in SMARCA4, with a similar phenotype of hearing impairment and abnormal bone formation in the auditory bullae in transgenic mice carrying the human mutation in the mouse SMARCA4 orthologue.


Asunto(s)
Pérdida Auditiva , Otosclerosis , Adulto , Humanos , Ratones , Animales , Otosclerosis/genética , Otosclerosis/cirugía , Vesícula/complicaciones , Estudio de Asociación del Genoma Completo , Reflejo de Sobresalto , Fenotipo , Ratones Transgénicos , Mutación , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética
3.
Cell Mol Life Sci ; 79(2): 113, 2022 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-35099616

RESUMEN

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Osteogénesis/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Células Cultivadas , Femenino , Células HEK293 , Humanos , Proteínas Relacionadas con Receptor de LDL/antagonistas & inhibidores , Proteínas Relacionadas con Receptor de LDL/química , Proteínas Relacionadas con Receptor de LDL/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteogénesis/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/genética , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes/química
4.
PLoS Biol ; 16(8): e2002979, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30142160

RESUMEN

There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and αvß3 integrin, which act in concert to facilitate bone resorption by osteoclasts. Using functional fluorescence-activated cell sorting (FACS)-based screening assays of random mutagenesis macrophage colony-stimulating factor (M-CSF) libraries against c-FMS and αvß3 integrin, we engineered dual-specific M-CSF mutants with high affinity to both receptors. These bispecific mutants act as functional antagonists of c-FMS and αvß3 integrin activation and hence of osteoclast differentiation in vitro and osteoclast activity in vivo. This study thus introduces a versatile platform for the creation of new-generation therapeutics with high efficacy and specificity for osteoporosis and other bone diseases. It also provides new tools for studying molecular mechanisms and the cell signaling pathways that mediate osteoclast differentiation and function.


Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/prevención & control , Integrina alfaVbeta3/antagonistas & inhibidores , Factor Estimulante de Colonias de Macrófagos/farmacología , Osteoclastos/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Animales , Sitios de Unión , Conservadores de la Densidad Ósea/química , Conservadores de la Densidad Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Factor Estimulante de Colonias de Macrófagos/química , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Simulación del Acoplamiento Molecular , Mutación , Osteoclastos/metabolismo , Osteoclastos/patología , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal
5.
PLoS Genet ; 14(10): e1007738, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30372441

RESUMEN

The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations.


Asunto(s)
Deformidades Congénitas de las Extremidades/genética , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/fisiología , Animales , Región Branquial/metabolismo , Elementos de Facilitación Genéticos/genética , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox , Histona Desacetilasas/genética , Proteínas de Homeodominio/genética , Esbozos de los Miembros/metabolismo , Deformidades Congénitas de las Extremidades/embriología , Ratones , Ratones Endogámicos C57BL , Organogénesis , Proteínas Represoras/genética , Factor de Transcripción AP-2 , Factores de Transcripción/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética
6.
J Oral Maxillofac Surg ; 78(12): 2226-2234, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32649899

RESUMEN

Cherubism is a rare autosomal dominant disease whose severity ranges widely, from asymptomatic to life-threatening. Bilateral symmetrical painless expansion of the mandible and maxilla resulting in a typical appearance of the face resembling a cherub, are the highlighted features of the condition. In most cases, cherubism-induced lesions in the jaws appear around the age of 3 years and tend to expand and increase in numbers until puberty. Treatment options for cherubism range from observation to surgical correction and various pharmacologic therapies. Given the excess sensitivity of cherubism osteoclasts to RANKL (receptor activator of nuclear factor κB ligand) and the positive effects of denosumab (XGEVA; Amgen, Thousand Oaks, CA) treatment in patients with giant cell granuloma, we have designed a treatment based on denosumab for 2 cherubism patients that achieves what we consider promising results.


Asunto(s)
Querubismo , Denosumab , Querubismo/tratamiento farmacológico , Denosumab/uso terapéutico , Humanos , Mandíbula , Maxilar , Pacientes
7.
FASEB J ; 32(5): 2507-2518, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29295862

RESUMEN

Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient ( Gpr39-/-) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39-/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39-/- mice. Furthermore, Gpr39-/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39-/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39-/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteoblasts and regulates collagen processing and deposition.-Jovanovic, M., Schmidt, F. N., Guterman-Ram, G., Khayyeri, H., Hiram-Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H., Busse, B., Jähn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.


Asunto(s)
Densidad Ósea , Matriz Ósea/metabolismo , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Animales , Matriz Ósea/patología , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/genética , Colágeno/biosíntesis , Colágeno/genética , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Receptores Acoplados a Proteínas G/metabolismo
8.
J Cell Physiol ; 233(1): 617-629, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28332708

RESUMEN

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign-body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion-founder cells that fuse to fusion-follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.


Asunto(s)
Fusión Celular , Osteoclastos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Resorción Ósea , Diferenciación Celular , Proliferación Celular , Regulación Enzimológica de la Expresión Génica , Células Gigantes de Cuerpo Extraño/enzimología , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Células RAW 264.7 , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Quinasas DyrK
9.
Biochem J ; 474(15): 2601-2617, 2017 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-28655719

RESUMEN

Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and overexpression of macrophage colony-stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essential for osteoclast differentiation, are known to promote bone metastasis and osteoporosis, making both the ligand and its receptor attractive targets for therapeutic intervention. With this aim in mind, our starting point was the previously held concept that the potential of the M-CSFC31S mutant as a therapeutic is derived from its inability to dimerize and hence to act as an agonist. The current study showed, however, that dimerization is not abolished in M-CSFC31S and that the protein retains agonistic activity toward osteoclasts. To design an M-CSF mutant with diminished dimerization capabilities, we solved the crystal structure of the M-CSFC31S dimer complex and used structure-based energy calculations to identify the residues responsible for its dimeric form. We then used that analysis to develop M-CSFC31S,M27R, a ligand-based, high-affinity antagonist for c-FMS that retained its binding ability but prevented the ligand dimerization that leads to receptor dimerization and activation. The monomeric properties of M-CSFC31S,M27R were validated using dynamic light scattering and small-angle X-ray scattering analyses. It was shown that this mutant is a functional inhibitor of M-CSF-dependent c-FMS activation and osteoclast differentiation in vitro Our study, therefore, provided insights into the sequence-structure-function relationships of the M-CSF/c-FMS interaction and of ligand/receptor tyrosine kinase interactions in general.


Asunto(s)
Sustitución de Aminoácidos , Diferenciación Celular/genética , Factor Estimulante de Colonias de Macrófagos , Mutación Missense , Multimerización de Proteína/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Animales , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Osteoclastos/citología , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Relación Estructura-Actividad
10.
J Biol Chem ; 290(43): 26180-93, 2015 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-26359491

RESUMEN

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF · c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF · c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF · c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.


Asunto(s)
Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Técnicas Químicas Combinatorias , Citometría de Flujo , Humanos , Factor Estimulante de Colonias de Macrófagos/química , Unión Proteica , Conformación Proteica
11.
Bone Res ; 10(1): 36, 2022 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-35396510

RESUMEN

Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α-/- mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

12.
Biomaterials ; 205: 11-22, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30901634

RESUMEN

Implementing ovarian tissue engineering for the maturation of primordial follicles, the most abundant follicle population in the ovary, holds great potential for women fertility preservation. Here, we evaluated whether macroporous alginate scaffolds with affinity-bound bone morphogenetic protein-4 (BMP-4) could mimic the ovary microenvironment and support the culture and growth of primordial follicles seeded with supporting ovarian cells. Porcine primordial follicles developed in the alginate scaffolds up to the pre-antral stage within 21 days. Affinity-bound BMP-4 significantly contributed to follicular maturation, as evident by the 5-fold increase in the number of developing follicles and enhanced estradiol secretion in these cultures compared to when BMP-4 was added to cultures with no affinity binding. After 21 days in culture, an increase in GDF-9/AMH gene expression, which is correlated with follicular development, was statistically significant when BMP-4 was affinity bound, compared to all other scaffold groups. When developed in-vivo, after xeno-transplantation of the follicle devices supplemented with additional angiogenic factors, the follicles reached antral size and secreted hormones at levels leading to restoration of ovarian function in ovariectomized severe combined immunodeficiency (SCID) mice. Altogether, our results provide first affirmation for the applicability of macroporous alginate scaffolds as a suitable platform for promoting follicle maturation in-vitro and in-vivo, and lay the foundations for the advantageous use of affinity binding presentation of growth factors to cultured follicles.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/farmacología , Ovario/efectos de los fármacos , Andamios del Tejido/química , Alginatos/farmacología , Animales , Proteína Morfogenética Ósea 4/farmacología , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas/sangre , Humanos , Ratones , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/crecimiento & desarrollo , Porosidad , Sulfatos/farmacología , Porcinos , Supervivencia Tisular/efectos de los fármacos
13.
Cell Calcium ; 75: 53-63, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30145429

RESUMEN

Zinc is an essential micronutrient affecting many aspects of human health. Cellular Zn2+ homeostasis is critical for cell function and survival. Zn2+, acting as a first or second messenger, triggers signaling pathways that mediate the physiological roles of Zn2+. Transient changes in Zn2+ concentrations within the cell or in the extracellular region occur following its release from Zn2+ binding metallothioneins, its transport across membranes by the ZnT or ZIP transporters, or release of vesicular Zn2+. These transients activate a distinct Zn2+ sensing receptor, ZnR/GPR39, or modulate numerous proteins and signaling pathways. Importantly, Zn2+ signaling regulates cellular physiological functions such as: proliferation, differentiation, ion transport and secretion. Indeed, novel therapeutic approaches aimed to maintain Zn2+ homeostasis and signaling are evolving. This review focuses on recent findings describing roles of Zn2+ and its transporters in regulating physiological or pathological processes.


Asunto(s)
Señalización del Calcio , Zinc/metabolismo , Animales , Enfermedad , Homeostasis , Humanos , Proteínas de Transporte de Membrana/metabolismo , Zinc/deficiencia
14.
Sci Rep ; 8(1): 13005, 2018 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-30158544

RESUMEN

Osteoclasts are the bone resorbing cells that derive from myeloid progenitor cells. Although there have been recent advancements in the ability to identify osteoclast progenitors, very little is known about the molecular mechanisms governing their homeostasis. Here, by analyzing the normalized phylogenetic profiles of the Schlafen (Slfn) gene family, we found that it co-evolved with osteoclast-related genes. Following these findings, we used a Slfn2 loss-of-function mutant mouse, elektra, to study the direct role of Slfn2 in osteoclast development and function. Slfn2eka/eka mice exhibited a profound increase in their cancellous bone mass and a significant reduction in osteoclast numbers. In addition, monocyte cultures from the bone marrow of Slfn2eka/eka mice showed a reduction in osteoclast number and total resorption area. Finally, we show that the bone marrow of Slfn2eka/eka mice have significantly less CD11b-Ly6Chi osteoclast precursors. Overall, our data suggest that Slfn2 is required for normal osteoclast differentiation and that loss of its function in mice results in an osteopetrotic phenotype.


Asunto(s)
Proteínas de Ciclo Celular/deficiencia , Diferenciación Celular , Mutación , Células Progenitoras Mieloides/fisiología , Osteoclastos/fisiología , Osteopetrosis/genética , Osteopetrosis/patología , Animales , Ratones
16.
Bone ; 79: 21-8, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26008608

RESUMEN

Osteoclasts are multinucleated, bone-resorbing cells formed via fusion of monocyte progenitors, a process triggered by prolonged stimulation with RANKL, the osteoclast master regulator cytokine. Monocyte fusion into osteoclasts has been shown to play a key role in bone remodeling and homeostasis; therefore, aberrant fusion may be involved in a variety of bone diseases. Indeed, research in the last decade has led to the discovery of genes regulating osteoclast fusion; yet the basic cellular regulatory mechanism underlying the fusion process is poorly understood. Here, we applied a novel approach for tracking the fusion processes, using live-cell imaging of RANKL-stimulated and non-stimulated progenitor monocytes differentially expressing dsRED or GFP, respectively. We show that osteoclast fusion is initiated by a small (~2.4%) subset of precursors, termed "fusion founders", capable of fusing either with other founders or with non-stimulated progenitors (fusion followers), which alone, are unable to initiate fusion. Careful examination indicates that the fusion between a founder and a follower cell consists of two distinct phases: an initial pairing of the two cells, typically lasting 5-35 min, during which the cells nevertheless maintain their initial morphology; and the fusion event itself. Interestingly, during the initial pre-fusion phase, a transfer of the fluorescent reporter proteins from nucleus to nucleus was noticed, suggesting crosstalk between the founder and follower progenitors via the cytoplasm that might directly affect the fusion process, as well as overall transcriptional regulation in the developing heterokaryon.


Asunto(s)
Monocitos/citología , Osteoclastos/citología , Ligando RANK/metabolismo , Células Madre/citología , Animales , Resorción Ósea/metabolismo , Diferenciación Celular/fisiología , Fusión Celular , Línea Celular , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Ratones , Monocitos/metabolismo , Osteoclastos/metabolismo , Células RAW 264.7 , Células Madre/metabolismo
17.
J Clin Invest ; 121(8): 3244-57, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21765218

RESUMEN

A fine balance between bone resorption by osteoclasts and bone formation by osteoblasts maintains bone homeostasis. In patients with cherubism, gain-of-function mutations in 3BP2, which is encoded by SH3-domain binding protein 2 (SH3BP2), cause cystic lesions with activated osteoclasts that lead to craniofacial abnormalities. However, little is known about the function of wild-type 3BP2 in regulating bone homeostasis. Here we have shown that 3BP2 is required for the normal function of both osteoblasts and osteoclasts. Initial analysis showed that Sh3bp2-/-mice developed osteoporosis as a result of reduced bone formation despite the fact that bone resorption was impaired. We demonstrated using reciprocal bone marrow chimeras, a cell-intrinsic defect of the osteoblast and osteoclast compartments in vivo. Further, Sh3bp2-/- osteoblasts failed to mature and form mineralized nodules in vitro, while Sh3bp2-/- osteoclasts spread poorly and were unable to effectively degrade dentine matrix in vitro. Finally, we showed that 3BP2 was required for Abl activation in osteoblasts and Src activation in osteoclasts, and demonstrated that the in vitro defect of each cell type was restored by the respective expression of activated forms of these kinases. These findings reveal an unanticipated role for the 3BP2 adapter protein in osteoblast function and in coordinating bone homeostatic signals in both osteoclast and osteoblast lineages.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica , Osteoclastos/metabolismo , Osteoporosis/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Médula Ósea/metabolismo , Resorción Ósea , Linaje de la Célula , Integrinas , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Osteoblastos/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo
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